Bovine Adeno-Associated Viral (BAAV) Vector and Uses Thereof

ABSTRACT

The present invention provides a bovine adeno-associated virus (BAAV) virus and vectors and particles derived therefrom. In addition, the present invention provides methods of delivering a nucleic acid to a cell using the BAAV vectors and particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/413,321, filed Mar. 6, 2012; which is a divisional of U.S. patentapplication Ser. No. 10/581,228, filed Oct. 26, 2006; which is a 371national phase filing of PCT/US04/40825, filed Dec. 6, 2004; whichclaims the benefit of U.S. Provisional Application No. 60/526,786, filedDec. 4, 2003; all of which are entitled, “Bovine Adeno-Associated Viral(BAAV) Vector and Uses Thereof”; and which also claims the benefit ofU.S. Provisional Application No. 60/607,854 filed Sep. 8, 2004, entitled“Trancytosis of Adeno-Associated Viruses”; all of which are herebyincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides bovine adeno-associated virus (BAAV) andvectors derived therefrom. Thus, the present invention relates to BAAVvectors for and methods of delivering nucleic acids to cells ofsubjects.

2. Background Art

Adeno-associated virus (AAV) is a member of the Parvoviridae, a virusfamily characterized by a single stranded linear DNA genome and a smallicosahedral shaped capsid measuring about 20 nm in diameter. AAV wasfirst described as a contamination of tissue culture grown simian virus15, a simian adenovirus and was found dependent on adenovirus formeasurable replication. This lead to its name, adeno-associated virus,and its classification in the genus Dependovirus (reviewed in Hoggan etal., 1970). AAV is a common contaminant of adenovirus samples and hasbeen isolated from human virus samples (AAV2, AAV3, AAV5), from samplesof simian virus-15 infected cells (AAV1, AAV4) as well as from stocks ofavian (AAAV) (Bossis and Chiorini, 2003), bovine, canine and ovineadenovirus and laboratory adenovirus type 5 stock (AAV6). DNA spanningthe entire rep-cap ORFs of AAV7 and AAV8 was amplified by PCR from hearttissue of rhesus monkeys (Gao et al., 2002).

With the exception of AAVs 1 and 6, all cloned AAV isolates appear to beserologically distinct. Nine isolates have been cloned, and recombinantviral stocks have been generated from each isolated virus.

AAV appears to commonly infect humans. 50%-80% of adults in NorthAmerica are seropositive for AAV. A steep rise in antibody responseagainst AAV 1-3 was observed in the age group between 1-10 years(Blacklow et al., 1968). AAV 2 and 3 were readily isolated from anal andthroat specimens from children (Blacklow et al., 1967) whereas isolationfrom adults was not observed. It appears that AAV spreads primarily inthe young population (Hoggan, 1970). Prevalence of antibodies againstAAV was found to be similar in Europe, Brazil and Japan, which suggestsa global spread of AAV (Erles et al., 1999). Infection with AAV appearsto be benign in man and laboratory animals. Currently, no disease hasbeen associated with AAV infections.

AAV2 is the best characterized adeno-associated virus and will bediscussed as an AAV prototype. The AAV2 genome consists of a linearsingle stranded DNA of 4,780 nucleotides. Both polarities of DNA areencapsulated by AAV with equal efficiency. The AAV2 genome contains 2open reading frames (ORF) named rep and cap. The rep ORF encodes thenon-structural proteins that are essential for viral DNA replication,packaging and AAV integration. The cap ORF encodes the capsid proteins.The rep ORF is transcribed from promoters at map units P5 and P19. Therep transcripts contain an intron close to the 3′ end of the rep ORF andcan be alternatively spliced. The rep ORF is therefore expressed as 4partially overlapping proteins, which were termed according to theirmolecular weight Rep78, 68, 52 and 40. The cap ORF is expressed from asingle promoter at P40. By alternative splicing and utilization of analternative ACG start codon, cap is expressed into the capsid proteinsVP1-3 which range in size from 65-86 kDa. VP3 is the most abundantcapsid protein and constitutes 80% of the AAV2 capsid. All viraltranscripts terminate at a polyA signal at map unit 96.

During a productive AAV2 infection, unspliced mRNAs from the p5 promoterencoding Rep78 are the first detectable viral transcripts. In the courseof infection, expression from P5, P19 and P40 increase to 1:3:18 levelsrespectively. The levels of spliced transcripts increased to 50% for P5,P19 products and 90% of P40 expressed RNA (Mouw and Pintel, 2000).

The AAV2 genome is terminated on both sides by inverted terminal repeats(ITRs) of 145 nucleotides (nt). 125 nt of the ITR constitute apalindrome which contains 2 internal palindromes of 21 nt each. The ITRcan fold back on itself to generate a T-shaped hairpin with only 7non-paired bases. The stem of the ITR contains a Rep binding site (RBS)and a sequence that is site and strand specifically cleaved by Rep—theterminal resolution site (TRS). The ITR is essential for AAV2 genomereplication, integration and contains the packaging signals.

The single-stranded AAV2 genome is packaged into a non-envelopedicosahedral shaped capsid of about 20-25 nm diameter. The virionconsists of 26% DNA and 74% protein and has a density of 1.41 g/cm³.AAV2 particles are extremely stable and can withstand heating to 60° C.for 1 hour, extreme ph, and extraction with organic solvents.

Rep proteins are involved in almost every step of AAV2 replicationincluding AAV2 genome replication, integration, and packaging. Rep78 andRep68 possess ATPase, 3′-5′ helicase, ligase and nicking activities andbind specifically to DNA. Rep52 and Rep40 appear to be involved in theencapsidation process and encode ATPase and 3′-5′ helicase activities.Mutational analysis suggests a domain structure for Rep78. TheN-terminal 225 aa are involved in DNA binding, DNA nicking and ligation.Rep78 and Rep68 recognize a GCTC repeat motif in the ITR as well as in alinear truncated form of the ITR (Chiorini et al., 1994) with similarefficiencies. Rep78 and Rep68 possess a sequence and strand specificendonuclease activity, which cleaves the ITR at the terminal resolutionsite (TRS). Rep endonuclease activity is dependent on nucleosidetriphosphate hydrolysis and presence of metal cations. Rep 78 and 68 canalso bind and cleave single stranded DNA in a NTP independent matter. Inaddition, Rep78 catalyzes rejoining of single stranded DNA substratesoriginating from the AAV2 origin of replication—i.e., sequencescontaining a rep binding and terminal resolution element.

The central region of AAV2 Rep78, which represents the N-terminus ofRep52 and Rep40, contains the ATPase and 3′-5′ helicase activities aswell as nuclear localization signals. The helicase activity unwindsDNA-DNA and DNA-RNA duplexes, but not RNA-RNA. The ATPase activity isconstitutive and independent of a DNA substrate. The C-terminus of Rep78contains a potential zinc-finger domain and can inhibit the cellularserine/threonine kinase activity of PKA as well as its homolog PRKX bypseudosubstrate inhibition. Rep68 which is translated from a splicedmRNA that encodes the N-terminal 529 amino acids (aa) of Rep78 fused to7 aa unique for Rep68, doesn't inhibit either PKA or PRKX. In additionto these biochemical activities, Rep can affect intracellular conditionsby protein-protein interactions. Rep78 binds to a variety of cellularproteins including transcription factors like SP-1, high-mobility-groupnon-histone protein 1 (HMG-1) and the oncosuppressor p53. Overexpressionof Rep results in pleiotrophic effects. Rep78 disrupts cell cycleprogression and inhibits transformation by cellular and viral oncogenes.In susceptible cell lines, overexpression of Rep resulted in apoptosisand cell death. Several of Rep78 activities contribute to cytotoxicity,including its constitutive ATPase activity, interference with cellulargene expression and protein interactions.

The first step of an AAV infection is binding to the cell surface.Receptors and coreceptors for AAV2 include heparan sulfate proteoglycan,fibroblast growth factor receptor-1, and α_(v)β₅ integrins whereasN-linked 2,3-linked sialic acid is required for AAV5 binding andtransduction (Walters et al., 2001). In HeLa cells, fluorescentlylabeled AAV2 particles appear to enter the cell via receptor-mediatedendocytosis in clathrin coated pits. More than 60% of bound virus wasinternalized within 10 min after infection. Labeled AAV particles areobserved to have escaped from the endosome, been trafficked via thecytoplasm to the cell nucleus and accumulated perinuclear, beforeentering the nucleus, probably via nuclear pore complex (NPC). AAV2particles have been detected in the nucleus, suggesting that uncoatingtakes place in the nucleus (Bartlett et al., 2000; Sanlioglu et al.,2000). AAV5 is internalized in HeLa cells predominantly by clathrincoated vesicles, but to a lesser degree also in noncoated pits. AAVparticles can also be trafficked intercellularly via the Golgi apparatus(Bantel-Schaal et al., 2002). At least partial uncoating of AAV5 wassuggested to take place before entering the nucleus since intact AAV5particles could not be detected in the nucleus (Bantel-Schaal et al.,2002) After uncoating, the single stranded genome is converted intoduplex DNA either by leading strand synthesis or annealing of input DNAof opposite polarity. AAV replication takes place within the nucleus.

During a co-infection with a helper virus such as Adenovirus, herpessimplex virus or cytomegalovirus, AAV is capable of an efficientproductive replication. The helper functions provided by Adenovirus havebeen studied in great detail. In human embryonic kidney 293 cells, whichconstitutively express the Adenovirus E1A and E1B genes, the earlyAdenovirus gene products of E2A, E4 and VA were found sufficient toallow replication of recombinant AAV. Allen et al. reported thatefficient production of rAAV is possible in 293 cells transfected withonly an E4orf6 expression plasmid (Allen et al., 2000). E1A stimulates Sphase entry and induces unscheduled DNA synthesis by inactivating thepRB checkpoint at the G1/S border by interaction with pRB familyproteins which results in the release of E2F (reviewed in (Ben-Israeland Kleinberger, 2002). This leads to either induction or activation ofenzymes involved in nucleotide synthesis and DNA replication. Sinceunscheduled DNA synthesis is a strong apoptotic signal, anti-apoptoticfunctions are required. E1B-19k is a Bcl-2 homolog and E1B-55k is a p53antagonist. Both proteins have anti-apoptotic functions. E4orf6 forms acomplex with E1B-55k and results in degradation of p53. It is alsoreported to cause S-phase arrest (Ben-Israel and Kleinberger, 2002). E2Aencodes a single strand DNA binding protein, which appears to benon-essential for DNA replication but effects gene expression (Chang andShenk, 1990) (Fields 39, 40). The VA transcription unit affects AAV2 RNAstability and translation (Janik et al., 1989). E1A has a more directeffect on AAV2 gene expression. The cellular transcription factor YY-1binds and inhibits the viral P5 promoter. E1A relieves thistranscriptional block. None of the late Ad gene products have been foundto be essential for AAV2 replication. The main function of the helpervirus appears to be the generation of a cellular environment with activeDNA replication machinery and blocked pro-apoptotic functions thatallows high-level AAV replication rather than a direct involvement inAAV replication.

While AAV is usually dependent on a helper virus for efficientreplication, low level AAV replication was observed under conditions ofgenotoxic stress (Yakinoglu et al., 1988; Yakobson et al., 1989). AAVDNA replication and particle formation was also observed indifferentiating keratinocytes in the absence of helper virus infection(Meyers et al., 2000). This demonstrates that AAV is not defective perse but rather depends on the helper virus to establish the favorablecellular condition and to provide factors for efficient replication

The ability of AAV vectors to infect dividing and non-dividing cells,establish long-term transgene expression, and the lack of pathogenicityhas made them attractive for use in gene therapy applications. Lack ofcross competition in binding experiments suggests that each AAV serotypemay have a distinct mechanism of cell entry. Comparison of the cap ORFsfrom different serotypes has identified blocks of conserved anddivergent sequence, with most of the latter residing on the exterior ofthe virion, thus explaining the altered tissue tropism among serotypes(19-21, 48, 56). Vectors based on new AAV serotypes may have differenthost range and different immunological properties, thus allowing formore efficient transduction in certain cell types. In addition,characterization of new serotypes will aid in identifying viral elementsrequired for altered tissue tropism.

Hearing and balance depend on the function of inner ear sensoryepithelia, which consists of hair cells and a number of supporting cellsthat provide mechanical support for the sensory cells. The developmentof efficient transgene delivery for the inner ear is an important steptowards potential application of gene-based therapies for cochleardisorders. Recently, a number of genes implicated in inheritedperipheral hearing and vestibular disorders that affect specific celltypes have been described. For example, a mutation of espin causesstereocilia degeneration (Naz, S., et al. J Med Genet. 2004 August;41(8):591-5), while mutations in connexins disrupt junctions betweensupporting cells (Kelsell, D. P., et al. Nature. 1997 May 1;387(6628):80-3), these references herein incorporated by reference forthe teaching of these mutations.

Some hereditary hearing loss disorders as well as progressive forms ofdeafness such as age-related hearing loss comprise excellent targets forgene therapy.

Currently, methods for introducing transgenes into neuroepithelial cellsin the inner ear are unsatisfactory. Several gene transfer vectorsincluding adeno-, lenti-, herpes simplex, and adenoassociated virus werecharacterized both in vivo and in vitro using cultured inner ear sensoryepithelia explants. While promising, each system had limitationsconcerning transduction efficiency, tropism, or non-specific pathologyinduced by the vector (Holt 2002)(Derby, Sena-Esteves et al. 1999; Holt,Johns et al. 1999). Conventional transfection methods using cationiclipids, DEAE-Dextran or calcium phosphate or electroporation are noteffective in inner ear epithelia and cause tissue degeneration.Transgenes may be introduced into sensory and nonsensory cells using aGene Gun™, where plasmids precipitated on gold carriers are introducedinto cells using high-pressure helium. While this approach offers theadvantage of rapid and simultaneous gene expression in all transfectedcells, and the ability to use easily manipulated plasmid DNA's, theextremely low yield of transfection as well as nonspecific structuraldamage to epithelia restricts its utility.

Provided is a vector comprising the BAAV virus or a vector comprisingsubparts of the virus, as well as BAAV viral particles. While BAAV issimilar to AAV1-8, the viruses are found herein to be physically andgenetically distinct. These differences endow BAAV with some uniqueproperties and advantages, which better suit it as a vector for genetherapy or gene transfer applications.

As shown herein, BAAV capsid proteins are distinct from primate andavian AAV capsid proteins and BAAV exhibits a distinct cell tropism,thus making BAAV capsid-containing particles suitable for transducingcell types for which primate or avian recombinant AAV particles areunsuited or less well-suited. BAAV is serologically distinct from otherAAVs and humans are not reported to have neutralizing antibodies againstBAAV, thus in a gene therapy application, BAAV would allow fortransduction of a patient who already possesses neutralizing antibodiesto primate isolates either as a result of natural immunological defenseor from prior exposure to other vectors. Thus, by providing these newrecombinant vectors and particles based on BAAV, a new and highly usefulseries of vectors and methods of using them are provided.

SUMMARY OF THE INVENTION

A nucleic acid vector comprising a pair of bovine adeno-associated virus(BAAV) inverted terminal repeats and a promoter between the invertedterminal repeats is provided.

Further provided is a BAAV particle containing a vector comprising apair of BAAV inverted terminal repeats.

Further provided is a BAAV particle containing a vector comprising apair of AAV 1 inverted terminal repeats.

Further provided is a BAAV particle containing a vector comprising apair of AAV2 inverted terminal repeats.

Further provided is a BAAV particle containing a vector comprising apair of AAV3 inverted terminal repeats.

Further provided is a BAAV particle containing a vector comprising apair of AAV4 inverted terminal repeats.

Further provided is a BAAV particle containing a vector comprising apair of AAV5 inverted terminal repeats.

Further provided is a BAAV particle containing a vector comprising apair of AAV6 inverted terminal repeats.

Further provided is a BAAV particle containing a vector comprising apair of AAV7 inverted terminal repeats.

Further provided is a BAAV particle containing a vector comprising apair of AAV8 inverted terminal repeats.

Further provided is a BAAV particle containing a vector comprising apair of AAAV inverted terminal repeats.

Further provided is a BAAV particle containing a vector comprising apair of AAV5 inverted terminal repeats.

Further provided is an AAV1 particle containing a vector comprising apair of BAAV inverted terminal repeats.

Further provided is an AAV2 particle containing a vector comprising apair of BAAV inverted terminal repeats.

Further provided is an AAV3 particle containing a vector comprising apair of BAAV inverted terminal repeats.

Further provided is an AAV4 particle containing a vector comprising apair of BAAV inverted terminal repeats.

Further provided is an AAV5 particle containing a vector comprising apair of BAAV inverted terminal repeats.

Further provided is an AAV6 particle containing a vector comprising apair of BAAV inverted terminal repeats.

Further provided is an AAV7 particle containing a vector comprising apair of BAAV inverted terminal repeats.

Further provided is an AAV8 particle containing a vector comprising apair of BAAV inverted terminal repeats.

Further provided is an AAAV particle containing a vector comprising apair of BAAV inverted terminal repeats.

Further provided is a dependovirus particle containing a vectorcomprising a pair of BAAV inverted terminal repeats.

Additionally, provided is an isolated nucleic acid comprising thenucleotide sequence set forth in SEQ ID NO:1 (BAAV genome). Furthermore,provided is an isolated nucleic acid consisting essentially of thenucleotide sequence set forth in SEQ ID NO:1 (BAAV genome).

Provided is an isolated nucleic acid encoding a BAAV Rep78 protein, forexample, the nucleic acid as set forth in SEQ ID NO:2. Additionallyprovided is an isolated full-length BAAV Rep78 protein as set forth inSEQ ID NO:3 or a unique fragment thereof. Additionally, provided is anisolated BAAV Rep 52 protein encoded by nucleic acid as set forth in SEQID NO:4 having the amino acid sequence set forth in SEQ ID NO:5, or aunique fragment thereof. The sequences for these proteins as well as thenucleotide sequence of the corresponding open reading frames areprovided below in the Sequence Listing and elsewhere in the applicationwhere the proteins are described.

Further provided is an isolated BAAV capsid protein, VP1, encoded bynucleic acid as set forth in SEQ ID NO:6 having the amino acid sequenceset forth in SEQ ID NO:7, or a unique fragment thereof. Additionallyprovided is an isolated BAAV capsid protein, VP2, encoded by nucleicacid as set forth in SEQ ID NO:8 having the amino acid sequence setforth in SEQ ID NO:9, or a unique fragment thereof. Also provided is anisolated BAAV capsid protein, VP3, encoded by nucleic acid as set forthin SEQ ID NO:10 having the amino acid sequence set forth in SEQ IDNO:11, or a unique fragment thereof.

Additionally provided is an isolated nucleic acid comprising a BAAV p5promoter having the nucleic acid sequence set forth in SEQ ID NO: 15, ora unique fragment thereof.

Provided is a method of screening a cell for infectivity by BAAVcomprising contacting the cell with BAAV and detecting the presence ofBAAV in the cells.

Further provided is a method of delivering a nucleic acid to a cellcomprising administering to the cell a BAAV particle containing a vectorcomprising the nucleic acid inserted between a pair of AAV invertedterminal repeats, thereby delivering the nucleic acid to the cell.

Further provided is a method of delivering a nucleic acid to a subjectcomprising administering to a cell from the subject a BAAV particlecomprising the nucleic acid inserted between a pair of AAV invertedterminal repeats, and returning the cell to the subject, therebydelivering the nucleic acid to the subject.

Further provided is a method of delivering a nucleic acid to a cell in asubject comprising administering to the subject a BAAV particlecomprising the nucleic acid inserted between a pair of AAV invertedterminal repeats, thereby delivering the nucleic acid to a cell in thesubject.

Further provided is a method of delivering a nucleic acid to a cell in asubject having antibodies to other serotypes of AAV comprisingadministering to the subject a BAAV particle comprising the nucleicacid, thereby delivering the nucleic acid to a cell in the subject.

Further provided is a BAAV particle comprising a capsid proteinconsisting essentially of the amino acid sequence set forth in SEQ IDNO:7, or a unique fragment thereof. Further provided is a BAAV particlecomprising a capsid protein consisting essentially of the amino acidsequence set forth in SEQ ID NO:9, or a unique fragment thereof. Furtherprovided is a BAAV particle comprising a capsid protein consistingessentially of the amino acid sequence set forth in SEQ ID NO:11, or aunique fragment thereof.

Additionally provided is an isolated nucleic acid comprising a BAAV p5promoter having the nucleic acid sequence set forth in SEQ ID NO:15, ora unique fragment thereof.

Provided is a method of screening a cell for infectivity by BAAV,comprising contacting the cell with BAAV and detecting the presence ofBAAV in the cells.

Further provided is a method of delivering a nucleic acid to a cellcomprising administering to the cell a BAAV particle containing a vectorcomprising the nucleic acid inserted between a pair of AAV invertedterminal repeats, thereby delivering the nucleic acid to the cell.

Further provided is a method of delivering a nucleic acid to a subjectcomprising administering to a cell from the subject a BAAV particlecomprising the nucleic acid inserted between a pair of BAAV invertedterminal repeats, and returning the cell to the subject, therebydelivering the nucleic acid to the subject.

Further provided is a method of delivering a nucleic acid to a cell in asubject comprising administering to the subject a BAAV particlecomprising the nucleic acid inserted between a pair of BAAV invertedterminal repeats, thereby delivering the nucleic acid to a cell in thesubject.

Further provided is a method of delivering a nucleic acid to a cell in asubject having antibodies to primate AAVs comprising administering tothe subject a BAAV particle comprising the nucleic acid, therebydelivering the nucleic acid to a cell in the subject.

Provided is a vector system for producing infectious virus particleshaving a characteristic of BAAV comprising: at least one vectorcomprising a nucleic acid selected from the group consisting of a pairof BAAV inverted terminal repeats, a nucleic acid encoding a BAAV capsidprotein, and a nucleic acid encoding a BAAV Rep protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show comparisons of the BAAV genome with other AAVgenomes. (A) The genomes of BAAV (SEQ ID NO:1), AAV2 (SEQ ID NO:26),AAV4 (SEQ ID NO:27), and AAV5 (SEQ ID NO:28), were aligned usingMACVECTOR™ (Oxford Molecular). Nucleotides identical in at least 2 AAVserotypes are displayed boxed and shaded. (B) Phylogenetic relationshipof BAAV to other serotypes is illustrated by an unrooted tree diagram.

FIG. 2 shows an example of a BAAV ITR (SEQ ID NO:29). The sequence ofthe ITR is shown in hairpin configuration. The putative Rep binding site(SEQ ID NO:24 and SEQ ID NO:25) and TRS element (SEQ ID NO:14) areboxed. Sequence changes relative to the AAV5 ITR are annotated eitherabove or below the BAAV sequence in bold letters.

FIGS. 3A-3D illustrate comparisons of Rep and VP1 amino acid sequences.(A) Alignment of the amino acid sequences of BAAV Rep protein, AAV2 Repprotein (SEQ ID NO:30) and AAV5 Rep protein (SEQ ID NO:31) usingMACVECTOR™. (B) Alignment of the amino acid sequences of BAAV VP1protein (SEQ ID NO:7), AAV2 VP1 protein (SEQ ID NO:32) and AAV4 VP1protein (SEQ ID NO:33) using MACVECTOR™. Identical amino acids areindicated by a dark, shaded box, similar amino acids by a light, shadedbox. Dashes indicate gaps in the sequence added by the alignmentprogram. Phylogenetic relationship of (C) BAAV Rep and (D) VP1 to otherAAV serotypes is illustrated by an unrooted tree diagram.

FIG. 4 shows the transduction profile of BAAV in 60 cancer cell lines.Human cell lines were infected with rBAAV expressing lacZ in serialdilutions and coinfected with a MOI of 10 with Ad5. Columns representbeta-Gal transducing units/10⁹ DNAse resistant rAAV particles.

FIGS. 5A-5B show that BAAV elicits a distinct immune response in mice.rBAAVlacZ (A) and rAAV4lacZ (B) were incubated with serial dilutions ofpolyclonal mice serum against rAAV2, rAAV4, rAAV5 and rBAAV. Cos cellscoinfected with Ad5 (MOI=10) were incubated with the virus/serummixture. % neutralization was calculated by the formula:100×(1−transducing titers of serum incubated rAAV/untreated rAAV).Values of neutralization that were calculated to be below zero wereadjusted to zero. Values given are means of 3 experiments, error barsrepresent standard deviation. BAAV transduction efficiency wasunaffected by antisera against AAV2, AAV4 and AAV5. Antisera againstBAAV blocked infection of BAAV but had no effect on the other AAVserotypes.

FIGS. 6A and 6B show a comparison of rAAV2 and rBAAV transduction ofsalivary glands. 10¹⁰ particles of AAV2-RnlacZ and BAAV-RnlacZ wereinjected into submandibular glands of BALB/c mice by retrograde ductalinstillation. 4 weeks after infection, glands were removed and analyzedfor the presence of vector genome DNA by real time PCR (A) andexpression of beta-gal by an ELISA (B). Values given are means of datafrom 7 animals, error bars represent standard deviation.

FIG. 7 is a comparison of transfection yield at increasing viral titers;At least 5 samples harvested from at least three explants were scoredfor transfected and not transfected hair cells at viral titers rangingfrom 10⁹ to 10¹¹ DRP/ml. The transfection yield increased significantlyfor OHC (N=5 and 13 Respectively, Single Factor ANOVA p=0.01699) and VHC(N=6 and 10 respectively, Single Factor ANOVA p=0.000168) with a 100fold increase in viral titer after 8 days. The differences intransfection yield of IHC were not significant (N=5 and 1, Single FactorANOVA p=0.23987).

FIGS. 8A-C show the apparent transfection efficiency increasedsignificantly with longer incubations with viral particles; A) Confocalimage of vestibular epithelia after 5 days of BAAV infection. Thepositively transduced hair cells are easily scored even though the yieldis sparse. B) Confocal image of vestibular epithelia after 8 days ofBAAV infection demonstrating almost half the hair cells are transfected.C) Histogram of transfection yields. There is a statisticallysignificant increase (N=7 and 10 frames respectively from at least 3explants p>0.01) in the transfected cells after an additional three daysof incubation. Size bar=20 um.

FIG. 9 is a comparison of the transfection efficiency of bovine AAV withthree primate AAVs. Cochlear and vestibular explants were incubated withvirus for 8 days and transfection yields were measured per sample frame.(A) BAAV transfected auditory and vestibular hair cells. (arrows) Incontrast AAV2, 4 and 5 were surprisingly ineffective. (arrows) (B) 10frames of each epithelia were measured and the transfection efficiencyscored. In general the primate derived adenoassociated viruses wereineffective as vectors for hair cell. Size bar=20 um.

FIG. 10 shows that BAAV transduction requires 2-3 sialic acid. Cos cellswere incubated with the broad spectrum neuraminidases isolated from V.cholerae, (0.05 U/ml) and a neuraminidase with high specificity for 2-3sialyl linkages from S. pneumoniae (10 U/ml). 48 h after infection withrecombinant AAV2, AAV4, AAV5, or BAAV expressing GFP, cells wereanalyzed for GFP expression. Neuraminidase treatment resulted inreduction of BAAV transduction, demonstrating the requirement for 2-3linked sialic acid, bound to either a protein or lipid receptor for BAAVtransduction.

FIG. 11 shows that BAAV transduction can be inhibited with inhibitors ofglycolipid synthesis. Untreated COS cells or cells incubated for 48 hwith 5 μM PPMP or 5 μM PDMP were infected with recombinant AAV2, AAV4,AAV5 or BAAV expressing GFP. 48 h after infection, cells were analyzedfor GFP expression. Inhibition of glycolipid synthesis resulted in clearreduction of rBAAV mediated gene transfer compared to untreated control,while rAAV2, rAAV4 and rAAV5 transduction was enhanced. This indicatedthe usage of phospholipids in rBAAV receptor binding or uptake.

FIG. 12 shows that the receptor for BAAV is protease resistant. Coscells were incubated with 0.025% trypsin or 1 U/ml dispase for 30 min.48 h after infection with recombinant AAV2, AAV4, AAV5 or BAAVexpressing GFP, cells were analyzed for GFP expression. Proteasetreatment resulted in reduction of rAAV2, rAAV4 and rAAV5 transduction,while BAAV mediated gene transfer was slightly enhanced, suggesting thateither a protease resistant protein or a lipid component is essentialfor rBAAV binding and uptake.

FIG. 13 shows that AAV4 transcytosed in CaCo-2, MDCKI, MDCKII, Humanprimary immortalized epithelial endometrial, Bovine brain primaryendothelia cells (BBB). AAV5 transcytosed CaCo-2 cells, whereas BAAVtranscytosed in MDCKs, Endometrial, airways epithelia, and BBB. AAV6 didnot transcytose in any of cell types tested. HeLa cells do not formbarrier epithelia and were used as a control.

FIG. 14 shows that the treatment of the basal lateral surface of Humanprimary airways epithelial cell (HAE) with tannic acid blocked thetranscytosis of BAAV vector containing a GFP expression cassette fromthe apical surface to the basal lateral. Furthermore transductiondramatically increased when assayed at 24 hrs post inoculation. Incontrast no change was observed in AAV2 transduction, which did notdemonstrate any transcytosis activity and has limited binding activityon HAE.

FIG. 15 shows that both AAV5 and BAAV efficiently transduce primaryairway epithelia cells. These cells were cultured and plated aspreviously described with an equivalent number of rAAV5 or rBAAVparticles containing CMV nuclear GFP—and cultured for over 10 days. GFPexpression was determined by flow cytometry (FACS) and the relativetransduction was compared.

DETAILED DESCRIPTION OF THE INVENTION

As used in the specification and in the claims, “a” can mean one ormore, depending upon the context in which it is used. The terms “having”and “comprising” are used interchangeably herein, and signify open endedmeaning.

The present application provides a recombinant bovine adeno-associatedvirus (BAAV). The application provides the isolation, subcloning, andsequencing of BAAV. This virus has one or more of the characteristicsdescribed below. The compositions provided herein do not includewild-type BAAV. The methods provided herein can use either wild-typeBAAV or recombinant BAAV-based delivery.

Provided herein are recombinant BAAV particles, recombinant BAAV vectorsand recombinant BAAV virions. As used herein, “recombinant” refers tonucleic acids, vectors, polypeptides, or proteins that have beengenerated using DNA recombination (cloning) methods and aredistinguishable from native or wild-type nucleic acids, vectors,polypeptides, or proteins. A BAAV particle is a viral particlecomprising a BAAV capsid protein. A recombinant BAAV vector is a nucleicacid construct that comprises at least one unique, isolated nucleic acidof BAAV. The recombinant BAAV vector can further comprise at least onenon-BAAV nucleic acid. As used herein, a “virion” refers to aninfectious virus particle, and “infectious” refers to the ability of avirion to deliver genetic material to a cell. Thus, a recombinant BAAVvirion is a particle containing a recombinant BAAV vector, wherein theparticle can be either a BAAV particle as described herein or a non-BAAVparticle. Alternatively, a recombinant BAAV virion can be a BAAVparticle containing a recombinant vector, wherein the vector can beeither a BAAV vector as described herein or a non-BAAV vector. A BAAVparticle can further be an “empty particle”, wherein the particle doesnot contain a nucleic acid, vector or plasmid, and is therefore notinfectious. These vectors, particles, virions, nucleic acids andpolypeptides are described below.

Provided herein is the nucleotide sequence of the BAAV genome andvectors and particles derived there from. Specifically, provided hereinis a nucleic acid vector, comprising a pair of BAAV inverted terminalrepeats (ITRs) and a promoter between the inverted terminal repeats. Therep proteins of AAV5 and BAAV will bind to the BAAV ITR and it canfunction as an origin of replication for packaging of recombinant AAVparticles. The minimum sequence necessary for this activity is the TRSsite where Rep cleaves in order to replicate the virus. Minormodifications in an ITR are contemplated and are those that will notinterfere with the hairpin structure formed by the ITR as describedherein and known in the art. Furthermore, to be considered within theterm it must retain the Rep binding site described herein.

The D− region of the AAV2 ITR, a single stranded region of the ITR,inboard of the TRS site, has been shown to bind a factor which dependingon its phosphorylation state correlates with the conversion of the AAVfrom a single stranded genome to a transcriptionally active form thatallows for expression of the viral DNA. This region is conserved betweenAAV2, 3, 4, and 6 but is divergent in AAV5 and BAAV (SEQ ID NO: 13). TheD+ region is the reverse complement of the D− region.

The promoter can be any desired promoter, selected by knownconsiderations, such as the level of expression of a nucleic acidfunctionally linked to the promoter and the cell type in which thevector is to be used. That is, the promoter can be tissue/cell-specific.Promoters can be prokaryotic, eukaryotic, fungal, nuclear,mitochondrial, viral or plant promoters. Promoters can be exogenous orendogenous to the cell type being transduced by the vector. Promoterscan include, for example, bacterial promoters, known strong promoterssuch as SV40 or the inducible metallothionein promoter, or an AAVpromoter, such as an AAV p5 promoter. Additionally, chimeric regulatorypromoters for targeted gene expression can be utilized. Examples ofthese regulatory systems, which are known in the art, include thetetracycline based regulatory system which utilizes the tettransactivator protein (tTA), a chimeric protein containing the VP 16activation domain fused to the tet repressor of Escherichia coli, theIPTG based regulatory system, the CID based regulatory system, and theEcdysone based regulatory system (No, D., et al., Proc Natl Acad SciUSA. 93(8):3346-3351 (1996)).

Other promoters include promoters derived from actin genes,immunoglobulin genes, cytomegalovirus (CMV), adenovirus, bovinepapilloma virus, adenoviral promoters, such as the adenoviral major latepromoter, an inducible heat shock promoter, respiratory syncytial virus,Rous sarcomas virus (RSV), etc., specifically, the promoter can be AAV2p5 promoter or AAV5 p5 promoter or BAAV p5 promoter. More specifically,the BAAV p5 promoter can be in about the same location in SEQ ID NO: 1as the AAV2 p5 promoter, in the corresponding AAV2 published sequence.Additionally, the p5 promoter may be enhanced by nucleotides 1-173 ofSEQ ID NO:1. Furthermore, smaller fragments of p5 promoter that retainpromoter activity can readily be determined by standard proceduresincluding, for example, constructing a series of deletions in the p5promoter, linking the deletion to a reporter gene, and determiningwhether the reporter gene is expressed, i.e., transcribed and/ortranslated. The promoter can be the promoter of any of the AAVserotypes, and can be the p19 promoter (SEQ ID NO: 16) or the p40promoter set forth in the sequence listing as SEQ ID NO: 17.

It should be recognized that any errors in any of the nucleotidesequences disclosed herein can be corrected, for example, by using thehybridization procedure described below with various probes derived fromthe described sequences such that the coding sequence can be re-isolatedand re-sequenced. Rapid screening for point mutations can also beachieved with the use of polymerase chain reaction-single strandconformation polymorphism (PCR-SSCP). The corresponding amino acidsequence can then be corrected accordingly.

The BAAV-derived vector provided herein can further comprise anexogenous nucleic acid functionally linked to the promoter. By“exogenous” nucleic acid is meant any nucleic acid that is not normallyfound in wild-type BAAV that can be inserted into a vector for transferinto a cell, tissue or organism. The exogenous nucleic acid can be anucleic acid not normally found in the target cell, or it can be anextra copy or copies of a nucleic acid normally found in the targetcell. The terms “exogenous” and “heterologous” are used hereininterchangeably.

By “functionally linked” is meant that the promoter can promoteexpression of the exogenous nucleic acid, as is known in the art, andcan include the appropriate orientation of the promoter relative to theexogenous nucleic acid. Furthermore, the exogenous nucleic acidpreferably has all appropriate sequences for expression of the nucleicacid. The nucleic acid can include, for example, expression controlsequences, such as an enhancer, and necessary information processingsites, such as ribosome binding sites, RNA splice sites, polyadenylationsites, and transcriptional terminator sequences.

The exogenous nucleic acid can encode beneficial proteins orpolypeptides that replace missing or defective proteins required by thecell or subject into which the vector is transferred or can encode acytotoxic polypeptide that can be directed, e.g., to cancer cells orother cells whose death would be beneficial to the subject. Theexogenous nucleic acid can also encode antisense RNAs that can bind to,and thereby inactivate, mRNAs made by the subject that encode harmfulproteins. The exogenous nucleic acid can also encode ribozymes that caneffect the sequence-specific inhibition of gene expression by thecleavage of mRNAs. In one aspect, antisense polynucleotides can beproduced from an exogenous expression cassette in an AAV5 vectorconstruct where the expression cassette contains a sequence thatpromotes cell-type specific expression (Wirak et al., EMBO 10:289(1991)). For general methods relating to antisense polynucleotides, seeAntisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1988).

Examples of exogenous nucleic acids which can be administered to a cellor subject as part of the present BAAV vector can include, but are notlimited to the following: nucleic acids encoding secretory andnonsecretory proteins, nucleic acids encoding therapeutic agents, suchas tumor necrosis factors (TNF), such as TNF-α; interferons, such asinterferon-α, interferon-β, and interferon-γ; interleukins, such asIL-1, IL-1β, and ILs -2 through -14; GM-CSF; adenosine deaminase;cellular growth factors, such as lymphokines; soluble CD4; Factor VIII;Factor IX; T-cell receptors; LDL receptor; ApoE; ApoC; alpha-1antitrypsin; ornithine transcarbamylase (OTC); cystic fibrosistransmembrane receptor (CFTR); insulin; Fc receptors for antigen bindingdomains of antibodies, such as immunoglobulins; anti-HIV decoy tarelements; and antisense sequences which inhibit viral replication, suchas antisense sequences which inhibit replication of hepatitis B orhepatitis non-A, non-B virus. The nucleic acid is chosen consideringseveral factors, including the cell to be transfected. Where the targetcell is a blood cell, for example, particularly useful nucleic acids touse are those which allow the blood cells to exert a therapeutic effect,such as a gene encoding a clotting factor for use in treatment ofhemophilia. Another target cell is the lung airway cell, which can beused to administer nucleic acids, such as those coding for the cysticfibrosis transmembrane receptor, which could provide a gene therapeutictreatment for cystic fibrosis. Other target cells include muscle cellswhere useful nucleic acids, such as those encoding cytokines and growthfactors, can be transduced and the protein the nucleic acid encodes canbe expressed and secreted to exert its effects on other cells, tissuesand organs, such as the liver. Furthermore, the nucleic acid can encodemore than one gene product, limited only, if the nucleic acid is to bepackaged in a capsid, by the size of nucleic acid that can be packaged.

Furthermore, suitable nucleic acids can include those that, whentransferred into a primary cell, such as a blood cell, cause thetransferred cell to target a site in the body where that cell's presencewould be beneficial. For example, blood cells such as TIL cells can bemodified, such as by transfer into the cell of a Fab portion of amonoclonal antibody, to recognize a selected antigen. Another examplewould be to introduce a nucleic acid that would target a therapeuticblood cell to tumor cells. Nucleic acids useful in treating cancer cellsinclude those encoding chemotactic factors which cause an inflammatoryresponse at a specific site, thereby having a therapeutic effect.

Cells, particularly blood cells, muscle cells, airway epithelial cells,brain cells and endothelial cells having such nucleic acids transferredinto them can be useful in a variety of diseases, syndromes andconditions. For example, suitable nucleic acids include nucleic acidsencoding soluble CD4, used in the treatment of AIDS and α-antitrypsin,used in the treatment of emphysema caused by α-antitrypsin deficiency.Other diseases, syndromes and conditions in which such cells can beuseful include, for example, adenosine deaminase deficiency, sickle celldeficiency, brain disorders such as Alzheimer's disease, thalassemia,hemophilia, diabetes, phenylketonuria, growth disorders and heartdiseases, such as those caused by alterations in cholesterol metabolism,and defects of the immune system.

Other cells in which a gene of interest can be expressed include, butare not limited to, fibroblasts, neurons, retinal cells, kidney cells,lung cells, bone marrow stem cells, hematopoietic stem cells, retinalcells and neurons. The cells in which the gene of interest can beexpressed can be dividing cells such as MDCK cells, BHK cells, HeLacells, 3T3 cells, CV1 cells, COST cells, HOS cells and 293 cells. Thecells can also be embryonic stem cells of mouse, rhesus, human, bovineor sheep origin, as well as stem cells of neural, hematopoietic, muscle,cardiac, immune or other origin. Non-dividing cells can also becontacted with a particle provided herein to express a gene of interest.Such cells include, but are not limited to hematopoietic stem cells andembryonic stem cells that have been rendered non-dividing.

As another example, hepatocytes can be transfected with the presentvectors having useful nucleic acids to treat liver disease. For example,a nucleic acid encoding OTC can be used to transfect hepatocytes (exvivo and returned to the liver or in vivo) to treat congenitalhyperammonemia, caused by an inherited deficiency in OTC. Anotherexample is to use a nucleic acid encoding LDL to target hepatocytes exvivo or in vivo to treat inherited LDL receptor deficiency. Suchtransfected hepatocytes can also be used to treat acquired infectiousdiseases, such as diseases resulting from a viral infection. Forexample, transduced hepatocyte precursors can be used to treat viralhepatitis, such as hepatitis B and non-A, non-B hepatitis, for exampleby transducing the hepatocyte precursor with a nucleic acid encoding anantisense RNA that inhibits viral replication. Another example includestransferring a vector provided herein having a nucleic acid encoding aprotein, such as γ-interferon, which can confer resistance to thehepatitis virus.

For a procedure using transfected hepatocytes or hepatocyte precursors,hepatocyte precursors having a vector provided herein transferred in canbe grown in tissue culture, removed from the tissue culture vessel, andintroduced to the body, such as by a surgical method. In this example,the tissue would be placed directly into the liver, or into the bodycavity in proximity to the liver, as in a transplant or graft.Alternatively, the cells can simply be directly injected into the liver,into the portal circulatory system, or into the spleen, from which thecells can be transported to the liver via the circulatory system.Furthermore, the cells can be attached to a support, such asmicrocarrier beads, which can then be introduced, such as by injection,into the peritoneal cavity. Once the cells are in the liver, by whatevermeans, the cells can then express the nucleic acid and/or differentiateinto mature hepatocytes which can express the nucleic acid.

The provided viral particles can be administered to cells, as describedherein, with a Multiplicity of Infection (MOI) of 10. The MOI is theratio of infectious virus particles to the number of cells beinginfected. Thus, an MOI of 0.1 results in the average inoculation of 1virus particle for every 10 cells. The general theory behind MOI is tointroduce one infectious virus particle to every host cell that ispresent in the culture.

However, more than one virus may infect the same cell which leaves apercentage of cells uninfected. This occurrence can be reduced by usinga higher MOI to ensure that every cell is infected. The provided viralparticles can therefore be administered to cells, as described herein,with a MOI of 0.01 to 100, such as for example 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80,90, 100.

The BAAV-derived vector can include any normally occurring BAAV nucleicacid sequences in addition to an ITR and promoter. The BAAV-derivedvector can also include sequences that are at least 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, or 99% identical to the BAAV nucleic acids set forthherein. Examples of vector constructs are provided below.

The present vector or BAAV particle or recombinant BAAV virion canutilize any unique fragment of these present BAAV nucleic acids,including the BAAV nucleic acids set forth in SEQ ID NOS: 1, 2, 4, 6, 8,10 and 12-17. A unique fragment consists of a sequence that is notpresent anywhere else on a genome. To be unique, the fragment must be ofsufficient size to distinguish it from other known sequences, which ismost readily determined by comparing any nucleic acid fragment to thenucleotide sequences of nucleic acids in computer databases, such asGenBank. Such comparative searches are standard in the art. Typically, aunique fragment useful as a primer or probe will be at least about 8 or10, preferable at least 20 or 25 nucleotides in length, depending uponthe specific nucleotide content of the sequence. Additionally, fragmentscan be, for example, at least about 30, 40, 50, 75, 100, 200 or 500nucleotides in length and can encode polypeptides or be probes. Thenucleic acid can be single or double stranded, depending upon thepurpose for which it is intended. Where desired, the nucleic acid can beRNA.

It is understood that as discussed herein the use of the terms“homology” and “identity” mean the same thing as similarity. Thus, forexample, if the use of the word homology is used to refer to twonon-natural sequences, it is understood that this is not necessarilyindicating an evolutionary relationship between these two sequences, butrather is looking at the similarity or relatedness between their nucleicacid sequences. Many of the methods for determining homology between twoevolutionarily related molecules are routinely applied to any two ormore nucleic acids or proteins for the purpose of measuring sequencesimilarity regardless of whether they are evolutionarily related.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed nucleicacids and polypeptides herein, is through defining the variants andderivatives in terms of homology to specific known sequences. Ingeneral, variants of nucleic acids and polypeptides herein disclosedtypically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99 percent homology to the stated sequence or the nativesequence. Those of skill in the art readily understand how to determinethe homology of two polypeptides or nucleic acids. For example, thehomology can be calculated after aligning the two sequences so that thehomology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.; theBLAST algorithm of Tatusova and Madden FEMS Microbiol. Lett. 174:247-250 (1999) available from the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989, which are herein incorporated byreference for at least material related to nucleic acid alignment. It isunderstood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

Further provided herein is a BAAV capsid protein that can combine withother capsid proteins to form a BAAV particle to contain the disclosedvectors. Also provided herein is a BAAV particle, comprising a BAAVcapsid protein. The capsid protein can be selected from a groupconsisting of VP1, VP2 and VP3. The capsid protein of the BAAV particlecan have the amino acid sequences of SEQ ID NOS: 7, 9, or 11. The capsidprotein of the BAAV particle can be encoded by the nucleic acidsequences of SEQ ID NOS: 6, 8, or 10. For example, provided is a BAAVparticle, comprising all three BAAV capsid proteins, i.e., VP1, VP2 andVP3, SEQ ID NOS: 7, 9, and 11, respectively. Also provided is a BAAVparticle, comprising each BAAV capsid protein individually or incombination. Also provided is a particle comprising VP1 and VP3 capsidproteins, i.e., lacking any VP2 capsid proteins. Thus, a BAAV particlecomprising a BAAV capsid protein comprises at least one BAAV capsidprotein (VP1, VP2 or VP3) or a functional fragment thereof. One of skillin the art understands that it is the non-conserved amino acids, asdemonstrated in FIG. 3, that are contributing to the properties of BAAVthat make it distinct from the other serotypes. Provided therefore is acapsid protein comprising a mutation, deletion or substitution in theconserved regions, including, for example, a substitution with ahomologous region from another AAV serotype.

A BAAV particle comprising a BAAV capsid protein can be utilized todeliver a nucleic acid vector to a cell, tissue or subject. For example,the herein described BAAV vectors can be encapsidated in a BAAVcapsid-derived particle and utilized in a gene delivery method.Furthermore, other viral nucleic acids can be encapsidated in the BAAVparticle and utilized in such delivery methods. For example, an AAV1-8or AAAV vector (e.g. AAV 1-8 or AAAV ITR and nucleic acid of interest)can be encapsidated in a BAAV particle and administered. Furthermore, aBAAV chimeric capsid incorporating AAV1-8 or AAAV capsid sequences andBAAV capsid sequences can be generated, by standard cloning methods,selecting regions from the known sequences of each protein as desired.For example, particularly antigenic regions of the BAAV capsid proteincan be replaced with the corresponding region of the AAV2 capsidprotein. In addition to chimeric capsids incorporating AAV2 capsidsequences, chimeric capsids incorporating AAV1, 3-8, and AAV5 capsidsequences can be generated, by standard cloning methods, selectingregions from the known sequences of each protein as desired.Alternatively a chimeric capsid can be made by the addition of a plasmidthat expresses AAV 1-8 capsid proteins at a ratio with the BAAV capsidexpression plasmid that allows only a few capsid proteins to beincorporated into the BAAV particle. Thus, for example, a chimericparticle may be constructed that contains 6 AAV2 capsid proteins and 54BAAV capsid proteins if the complete capsid contains 60 capsid proteins.Methods for generating chimeric AAVs are known in the art and can befound in Rabinowitz J E, et al. J Virol. 2004 May; 78(9):4421-32, hereinincorporated by reference for these methods. Examples of chimericcapsids would be to combine the VP1, 2, 3 proteins of BAAV and the VP1,2, 3 proteins of AAV5 such that a new tropism would arise. An examplewould be a vector that could both transduce and have transcytosisactivity in Caco-2 cells or a vector that could transduce a cell thatwas not previously permissive for either BAAV or AAV5.

The capsids can also be modified to alter their specific tropism bygenetically altering the capsid to encode a specific ligand to a cellsurface receptor.

Alternatively, the capsid can be chemically modified by conjugating aligand to a cell surface receptor. By genetically or chemically alteringthe capsids, the tropism can be modified to direct BAAV to a particularcell or population of cells. The capsids can also be alteredimmunologically by conjugating the capsid to an antibody that recognizesa specific protein on the target cell or population of cells.

Provided are two regions in the capsid of BAAV that are on the virussurface and could tolerate substitution. These two regions are aa257-264 (GSSNASDT SEQ ID NO:18) and aa 444-457 (TTSGGTLNQGNSAT SEQ IDNO:19). Other regions of the BAAV capsid could also accommodate thesubstitution of amino acids that would allow for epitope presentation onthe surface of the virus. All of these regions would have surfaceexposure and the ability to support a substitution of sequence to insertthe epitope while still allowing for capsid assembly. The substitutionscan include non-BAAV epitopes and non-BAAV ligands.

Because of the symmetry of the AAV particles, a substitution in onesubunit of the capsid will appear multiple times on the capsid surface.For example the capsid is made of approximately 50 VP3 proteins, 5 VP1and 5 VP2. Therefore an epitope incorporated in the VP3 protein could beexpressed 55 times on the surface of each particle increasing thelikelihood of the epitope forming a stable interaction with its target.In some cases this may be too high of a ligand density for functionalbinding or this high density of epitope may interfere with capsidformation. The epitope density could be lowered by introducing anotherplasmid into the packaging system for production of recombinantparticles and the ratio between the packaging plasmid with the modifiedVP3 protein and the wt VP3 protein altered to balance the epitopedensity on the virus surface. Thus, the ratio between the modified VP3and the wt VP3 can be 0:50 to 50:0, including, for example, 1:49, 2:48,3:47, 4:46, 5:45, 6:44, 7:43, 8:42, 9:41, 10:40, 11:39, 12:38, 13:37,14:36, 15:35, 16:34, 17:33, 18:32, 19:31, 20:30, 21:29, 22:28, 23:27,24:27, 25:25, 26:24, 27:23, 28:22, 29:21, 30:20, 31:19, 32:18, 33:17,34:16, 35:15, 36:14, 37:13, 38:12, 39:11, 40:10, 41:9, 42:8, 43:7, 44:6,45:5, 46:4, 47:3, 48:2, or 49:1.

Epitopes could be incorporated into the virus capsid for the purposeof 1) altering the tropism of the virus 2) blocking an immune responsedirected at the virus 3) developing a host immune response to theepitope for the purpose of vaccination. Examples of epitopes that couldbe added to BAAV capsids include but are not limited to:

LH receptor binding epitopeRGD integrin binding epitopeCD13 binding epitope NGRAHA SEQ ID NO:20The Retanef polyprotein vaccine candidate for HIV-1single chain antibody fragments directed against tumor cellsEndothelial cell binding epitope SIGYPLP SEQ ID NO:21serpin receptor ligand, KFNKPFVFLI SEQ ID NO:22protective B-cell epitope hemagglutinin (HA) 91-108 from influenza HANDV B-cell immunodominant epitope (IDE) spanning residues 447 to 455Major immunogenic epitope for parvovirus B19 (NISLDNPLENPSSLFDLVARIK SEQID NO:23) that can elicit protective antibody titers.

The capsids can also be assembled into empty particles by expression inmammalian, bacterial, fungal or insect cells. For example, AAV2particles are known to be made from VP3 and VP2 capsid proteins inbaculovirus. The same basic protocol can produce an empty BAAV particlecomprising BAAV capsid proteins and also full particles. The empty BAAVparticles can be used to deliver, for example, antigens, drugs,proteins, or metals to cells or cells in a subject. Antigens can bedirectly incorporated into the capsid of an empty BAAV particle. Anantigen can further be coupled via an antibody-antigen complex to theempty particle. Also disclosed is the coupling of drugs, proteins, ormetals on the inside of the empty particles.

The herein described recombinant BAAV nucleic acid derived vector can beencapsidated in a viral particle. The viral particle can be a parvovirusparticle. The parvovirus particle can be a dependovirus particle. Theviral particle can be an AAV particle. In particular, the recombinantBAAV nucleic acid derived vector can be encapsidated in a BAAV, AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAAV particle, a particlecomprising a portion of any of these capsids, or a chimeric capsidparticle as described above, by standard methods using the appropriatecapsid proteins in the encapsidation process, as long as the nucleicacid vector fits within the size limitation of the particle utilized.The encapsidation process itself is standard in the art. The BAAVreplication machinery, i.e. the rep initiator proteins and otherfunctions required for replication, can be utilized to produce the BAAVgenome that can be packaged in an AAV1-8 or AAAV capsid.

The recombinant BAAV virion containing a vector can also be produced byrecombinant methods utilizing multiple plasmids. In one example, theBAAV rep nucleic acid would be cloned into one plasmid, the BAAV ITRnucleic acid would be cloned into another plasmid and the AAV1-8 capsidnucleic acid would be cloned on another plasmid. These plasmids wouldthen be introduced into cells. The cells that were efficientlytransduced by all three plasmids, would exhibit specific integration aswell as the ability to produce BAAV recombinant virus. Additionally, twoplasmids could be used where the BAAV rep nucleic acid would be clonedinto one plasmid and the BAAV ITR and BAAV capsid would be cloned intoanother plasmid. These plasmids would then be introduced into cells. Thecells that were efficiently transduced by both plasmids, would exhibitspecific integration as well as the ability to produce BAAV recombinantvirus.

A BAAV capsid composed of VP1, VP2, and VP3 polypeptide can overall havegreater than 56% homology to the polypeptide having the amino acidsequence encoded by nucleotides in SEQ ID NOS:6, 8 and 10.

The capsid protein can have about 70% homology, about 75% homology, 80%homology, 85% homology, 90% homology, 95% homology, 98% homology, 99%homology, or even 100% homology to the protein having the amino acidsequence encoded by the nucleotides set forth in SEQ ID NOS:6, 8 or 10.The percent homology used to identify proteins herein, can be based on anucleotide-by-nucleotide comparison or more preferable is based on acomputerized algorithm as described herein. Variations in the amino acidsequence of the BAAV capsid protein are contemplated herein, as long asthe resulting particle comprising a BAAV capsid protein remainsantigenically or immunologically distinct from AAV1-8 or AAAV capsid, ascan be routinely determined by standard methods. Specifically, forexample, ELISA and Western blots can be used to determine whether aviral particle is antigenically or immunologically distinct from AAV2 orthe other serotypes. Furthermore, the BAAV particle preferably retainstissue tropism distinction from other AAVs, such as that exemplified inthe examples herein. A BAAV chimeric particle comprising at least oneBAAV coat protein may have a different tissue tropism from that of aBAAV particle consisting only of BAAV coat proteins, but is stilldistinct from the tropism of an AAV2 particle.

Provided herein is a recombinant BAAV virion, comprising a BAAV particlecontaining, i.e., encapsidating, a vector comprising a pair of AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAAV, or BAAV invertedterminal repeats. The recombinant vector can further comprise a BAAVRep-encoding nucleic acid. The vector encapsidated in the particle canfurther comprise an exogenous nucleic acid inserted between the invertedterminal repeats.

Further contemplated are chimeric recombinant ITRs that contain a repbinding site and a TRS site recognized by that Rep protein. By “Repprotein” is meant one or more of the Rep proteins, Rep 40, Rep 78, Rep52, Rep 68. Alternatively, “Rep protein” could be all four of the Repproteins described herein. One example of a chimeric ITR would consistof a BAAV D region (SEQ ID NO: 13), a BAAV TRS site (SEQ ID NO: 14), anAAV2 hairpin and an AAV2 Rep binding site. Another example would be aBAAV D region, a BAAV TRS site, an AAV3 hairpin and an AAV3 Rep bindingsite. In these chimeric ITRs, the D region can be from AAV1-8 or AAAV.The hairpin can be derived from AAV 1-8 or AAAV. The binding site can bederived from any of AAV1-8 or AAAV. Preferably, the D region and the TRSare from the same serotype.

The chimeric ITRs can be combined with BAAV Rep protein and any of theAAV serotype capsids to obtain a recombinant virion. For example, arecombinant virion can be produced by a BAAV D region, a BAAV TRS site,an AAV2 hairpin, an AAV2 binding site, BAAV Rep protein and AAV1 capsid.This recombinant virion would possess the cellular tropism conferred bythe AAV1 capsid protein and would possess the efficient replicationconferred by the BAAV Rep.

Other examples of the combinations of ITR, Rep protein and Capsids thatwill produce recombinant virus include but are not limited to:

BAAV ITR+BAAV Rep+BAAV Cap=virusAAV5 ITR+BAAV Rep+BAAV Cap=virusAAV5 ITR+BAAV Rep+AAV1 Cap=virusAAV5 ITR+BAAV Rep+AAV2 Cap=virusAAV5 ITR+BAAV Rep+AAV3 Cap=virusAAV5 ITR+BAAV Rep+AAV4 Cap=virusAAV5 ITR+BAAV Rep+AAV5 Cap=virusAAV5 ITR+BAAV Rep+AAV6 Cap=virusAAV5 ITR+BAAV Rep+AAV7 Cap=virusAAV5 ITR+BAAV Rep+AAV8 Cap=virusAAV5 ITR+BAAV Rep+AAAV Cap=virusBAAV ITR+AAV5 Rep+BAAV Cap=virusBAAV ITR+AAV5 Rep+AAV1 Cap=virusBAAV ITR+AAV5 Rep+AAV2 Cap=virusBAAV ITR+AAV5 Rep+AAV3 Cap=virusBAAV ITR+AAV5 Rep+AAV4 Cap=virusBAAV ITR+AAV5 Rep+AAV5 Cap=virusBAAV ITR+AAV5 Rep+AAV6 Cap=virusBAAV ITR+AAV5 Rep+AAV7 Cap=virusBAAV ITR+AAV5 Rep+AAV8 Cap=virusBAAV ITR+AAV5 Rep+AAAV Cap=virusAAV1 ITR+AAV1 Rep+BAAV Cap=virusAAV2 ITR+AAV2 Rep+BAAV Cap=virusAAV3 ITR+AAV3 Rep+BAAV Cap=virusAAV4 ITR+AAV4 Rep+BAAV Cap=virusAAV5 ITR+AAV5 Rep+BAAV Cap=virusAAV6 ITR+AAV6 Rep+BAAV Cap=virusAAV7 ITR+AAV7 Rep+BAAV Cap=virusAAV8 ITR+AAV8 Rep+BAAV Cap=virusAAAV ITR+AAAV Rep+BAAV Cap=virus

One of skill in the art would know how to employ standard techniques toobtain the sequences from any of AAV 1-8 or AAAV in order to combinethem with BAAV sequences. Examples of BAAV sequences that can beutilized in these constructs can be found herein and under GenBankAccession No. AY388617 and these sequences are hereby incorporated intheir entireties by this reference. Examples of AAV1 sequences that canbe utilized in these constructs can be found in GenBank under AccessionNo. AF063497 and these sequences are hereby incorporated in theirentireties by this reference. Examples of AAV2 sequences that can beutilized in these constructs can be found in GenBank under Accession No.AF043303 and these sequences are hereby incorporated in their entiretiesby this reference. Examples of AAV3 sequences that can be utilized inthese constructs can be found in GenBank under Accession No.NC_(—)001729 and these sequences are hereby incorporated in theirentireties by this reference. Examples of AAV4 sequences that can beutilized in these constructs can be found in GenBank under Accession No.U89790 and these sequences are hereby incorporated in their entiretiesby this reference. Examples of AAV5 sequences that can be utilized inthese constructs can be found in GenBank under Accession No. AF085716and these sequences are hereby incorporated in their entireties by thisreference. Examples of AAV6 sequences that can be utilized in theseconstructs can be found in GenBank under Accession No. NC_(—)001862 andAF028704 and these sequences are hereby incorporated in their entiretiesby this reference. Examples of AAV7 sequences that can be utilized inthese constructs can be found in GenBank under Accession No. AF513851and these sequences are hereby incorporated in their entireties by thisreference. Examples of AAV8 sequences that can be utilized in theseconstructs can be found in GenBank under Accession No. AF513852 andthese sequences are hereby incorporated in their entireties by thisreference. Examples of AAAV sequences that can be utilized in theseconstructs can be found in GenBank under Accession No. AY186198 andthese sequences are hereby incorporated in their entireties by thisreference.

In any of the constructs described herein, inclusion of a promoter ispreferred. As used in the constructs herein, unless otherwise specified,Cap (capsid) refers to any of BAAV VP1, BAAV VP2, BAAV VP3, combinationsthereof, functional fragments of any of VP1, VP2 or VP3, or chimericcapsids as described herein. The ITRs of the constructs describedherein, can be chimeric recombinant ITRs as described elsewhere in theapplication.

Conjugates of recombinant or wild-type BAAV virions and nucleic acids orproteins can be used to deliver those molecules to a cell. For example,the purified BAAV can be used as a vehicle for delivering DNA bound tothe exterior of the virus. Examples of this are to conjugate the DNA tothe virion by a bridge using poly-L-lysine or other charged molecule.Also contemplated are virosomes that contain BAAV structural proteins(BAAV capsid proteins), lipids such as DOTAP, and nucleic acids that arecomplexed via charge interaction to introduce DNA into cells.

Also provided herein are conjugates that utilize the BAAV capsid or aunique region of the BAAV capsid protein (e.g. VP1, VP2 or VP3 orcombinations thereof) to introduce DNA into cells. For example, the BAAVVP3 protein or fragment thereof, can be conjugated to a DNA on a plasmidthat is conjugated to a lipid. Cells can be infected using the targetingability of the VP3 capsid protein to achieve the desired tissue tropism,specific to BAAV. BAAV VP1 and VP2 proteins can also be utilized tointroduce DNA or other molecules into cells. By further incorporatingthe Rep protein and the AAV TRS into the DNA-containing conjugate, cellscan be transduced and targeted integration can be achieved. For example,if BAAV specific targeted integration is desired, a conjugate composedof the BAAV VP3 capsid, BAAV rep or a fragment of BAAV rep, BAAV TRS,the rep binding site, the exogenous DNA of interest, and a lipid, can beutilized to achieve BAAV specific tropism and BAAV specific targetedintegration in the genome.

Further provided herein are chimeric viruses where BAAV vectors can beencapsidated by herpes simplex virus (HSV) (Heister, T., et al. J Virol.2002 July; 76(14):7163-73), incorporated herein for its teaching ofHSV/AAV hybrid vectors), baculovirus or other viruses to achieve adesired tropism associated with another virus. For example, the BAAVITRs could be encapsidated by HSV and cells could be infected.Post-infection, the ITRs of BAAV could be acted on by BAAV rep providedin the system or in a separate vehicle to rescue BAAV from the genome.Therefore, the cellular tropism of HSV can be combined with BAAV repmediated targeted integration. Other viruses that could be utilized toconstruct chimeric viruses include lentivirus, retrovirus, pseudotypedretroviral vectors and adenoviral vectors.

Provided herein are isolated nucleic acids of BAAV. For example,provided is an isolated nucleic acid comprising the nucleotide sequenceset forth in SEQ ID NO:1 (BAAV genome). This nucleic acid, or portionsthereof, can be inserted into vectors, such as plasmids, yeastartificial chromosomes, or other viral vector (particle), if desired, bystandard cloning methods. Also provided is an isolated nucleic acidconsisting essentially of the nucleotide sequence set forth in SEQ IDNO:1.

The phrase “consisting essentially of” is used herein to refer to acomposition that comprises the essential characteristics of theidentified composition. By “essential” is meant the characteristics thatcontribute to the structure or function of the disclosed molecule. Thus,any substitution, deletion or addition to the provided composition thatdoes not significantly alter the defining characteristics of thecomposition are considered therein.

For example, if an amino acid sequence X is disclosed, then a providedpolypeptide consisting essentially of the amino acid sequence Xincludes, for example, conservative amino acid substitutions (asdescribed below) that do not significantly alter the essentialcharacteristics of the polypeptide, e.g., secondary/tertiary structureor function of the protein. The provided polypeptide can furtherconstitute a fusion protein or otherwise have additional N-terminal,C-terminal, or intermediate amino acid sequences, e.g., linkers or tags.“Linker”, as used herein, is an amino acid sequences or insertion thatcan be used to connect or separate two distinct polypeptides orpolypeptide fragments, wherein the linker does not otherwise contributeto the essential function of the composition. A polypeptide providedherein, can have an amino acid linker comprising, for example, the aminoacids GLS, ALS, or LLA. A “tag”, as used herein, refers to a distinctamino acid sequence that can be used to detect or purify the providedpolypeptide, wherein the tag does not otherwise contribute to theessential function of the composition. The provided polypeptide canfurther have deleted N-terminal, C-terminal or intermediate amino acidsthat do not contribute to the essential activity of the polypeptide.

As another example, if a nucleic acid X is disclosed, then a providednucleic acid consisting essentially of nucleic acid sequence X,includes, for example, nucleotide substitutions that do not alter theamino acid sequence of the encoded polypeptide, i.e., due to degeneracy.If sequence X comprises introns and exons, then the provided nucleicacid can further be the cDNA sequence that lacks the introns butcomprises the exons of sequence X. To the extent that specific geneswithin a genome are identified herein, it is further understood that thedisclosure of a nucleic acid consisting essentially of the genomesequence would include fragments of the genome such as isolatedsequences comprising a gene or genes within the genome.

Other characteristics of nucleic acid or amino acid sequences that arenot herein considered essential include, for example, junk DNA betweengenes or any identifiable sequence unit, e.g., promoters, enhancers,transmembrane domains, poly-adenylation sequences, signal sequences,etc., that when substituted or removed would be presumed by one skilledin the art to not significantly alter the essential characteristics ofthe disclosed sequence.

Thus, the nucleotides of SEQ ID NO:1 can have minor modifications andstill be contemplated herein. For example, modifications that do notalter the amino acid encoded by any given codon (such as by modificationof the third, “wobble,” position in a codon) can readily be made, andsuch alterations are known in the art. Furthermore, modifications thatcause a resulting neutral (conserved) amino acid substitution of asimilar amino acid can be made in a coding region of the genome.Additionally, modifications as described herein for the BAAV components,such as the ITRs, the p5 promoter, etc. are contemplated herein.Furthermore, modifications to regions of SEQ ID NO:1 other than in theITR, TRS, Rep binding site and hairpin are likely to be toleratedwithout serious impact on the function of the nucleic acid as arecombinant vector.

As used herein, the term “isolated” refers to a nucleic acid separatedor significantly free from at least some of the other components of thenaturally occurring organism, for example, the cell structuralcomponents or viral components commonly found associated with nucleicacids in the environment of the virus and/or other nucleic acids. Theisolation of the native nucleic acids can be accomplished, for example,by techniques such as cell lysis followed by phenol plus chloroformextraction, followed by ethanol precipitation of the nucleic acids. Thenucleic acids provided herein can be isolated from cells according toany of many methods well known in the art.

As used herein, the term “nucleic acid” refers to single- ormultiple-stranded molecules which may be DNA or RNA, or any combinationthereof, including modifications to those nucleic acids. The nucleicacid may represent a coding strand or its complement, or any combinationthereof. Nucleic acids may be identical in sequence to the sequenceswhich are naturally occurring for any of the genes discussed herein ormay include alternative codons which encode the same amino acid as thoseprovided herein, including that which is found in the naturallyoccurring sequence. These nucleic acids can also be modified from theirtypical structure. Such modifications include, but are not limited to,methylated nucleic acids, the substitution of a non-bridging oxygen onthe phosphate residue with either a sulfur (yielding phosphorothioatedeoxynucleotides), selenium (yielding phosphorselenoatedeoxynucleotides), or methyl groups (yielding methylphosphonatedeoxynucleotides).

Additionally provided is an isolated nucleic acid that selectivelyhybridizes with any nucleic acid disclosed herein, including the entireBAAV genome and any unique fragment thereof, including the Rep andcapsid encoding sequences, promoters and ITRs (e.g. SEQ ID NOS: 1, 2, 4,6, 8, 10, 12, 13, 14, 15, 16, 17). Specifically, the nucleic acid canselectively or specifically hybridize to an isolated nucleic acidconsisting of the nucleotide sequence set forth in SEQ ID NO:1 (BAAVgenome). Further provided is an isolated nucleic acid that selectivelyor specifically hybridizes with an isolated nucleic acid comprising thenucleotide sequence set forth in SEQ ID NO:1 (BAAV genome). By“selectively hybridizes” as used herein is meant a nucleic acid thathybridizes to one of the disclosed nucleic acids under sufficientstringency conditions without significant hybridization to a nucleicacid encoding an unrelated protein, and particularly, without detectablyhybridizing to nucleic acids of AAV2. Thus, a nucleic acid thatselectively hybridizes with a nucleic acid provided herein will notselectively hybridize under stringent conditions with a nucleic acidencoding a different protein or the corresponding protein from adifferent serotype of the virus, and vice versa. A “specificallyhybridizing” nucleic acid is one that hybridizes under stringentconditions to only a nucleic acid found in BAAV. Therefore, nucleicacids for use, for example, as primers and probes to detect or amplifythe target nucleic acids are contemplated herein. Nucleic acid fragmentsthat selectively hybridize to any given nucleic acid can be used, e.g.,as primers and or probes for further hybridization or for amplificationmethods (e.g., polymerase chain reaction (PCR), ligase chain reaction(LCR)). Additionally, for example, a primer or probe can be designedthat selectively hybridizes with both BAAV and a gene of interestcarried within the BAAV vector (i.e., a chimeric nucleic acid).

Stringency of hybridization is controlled by both temperature and saltconcentration of either or both of the hybridization and washing steps.Typically, the stringency of hybridization to achieve selectivehybridization involves hybridization in high ionic strength solution(6×SSC or 6×SSPE) at a temperature that is about 12-25° C. below theT_(m) (the melting temperature at which half of the molecules dissociatefrom their hybridization partners) followed by washing at a combinationof temperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the T_(m). The temperatureand salt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The washingtemperatures can be used as described above to achieve selectivestringency, as is known in the art. (Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987).A preferable stringent hybridization condition for a DNA:DNAhybridization can be at about 68° C. (in aqueous solution) in 6×SSC or6×SSPE followed by washing at 68° C. Stringency of hybridization andwashing, if desired, can be reduced accordingly as the degree ofcomplementarity desired is decreased, and further, depending upon theG-C or A-T richness of any area wherein variability is searched for.Likewise, stringency of hybridization and washing, if desired, can beincreased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

A nucleic acid that selectively hybridizes to any portion of the BAAVgenome is contemplated herein. Therefore, a nucleic acid thatselectively hybridizes to BAAV can be of longer length than the BAAVgenome, it can be about the same length as the BAAV genome or it can beshorter than the BAAV genome. The length of the nucleic acid is limitedon the shorter end of the size range only by its specificity forhybridization to BAAV, i.e., once it is too short, typically less thanabout 5 to 7 nucleotides in length, it will no longer bind specificallyto BAAV, but rather will hybridize to numerous background nucleic acids.Additionally contemplated herein is a nucleic acid that has a portionthat specifically hybridizes to BAAV and a portion that specificallyhybridizes to a gene of interest inserted within BAAV.

Provided is an isolated nucleic acid comprising a BAAV p5 promoter. Thenucleic acid can consist of the sequence set forth in SEQ ID NO:15. Thenucleic acid can consist essentially of the sequence set forth in SEQ IDNO:15. Further provided is a nucleic acid that selectively hybridizeswith the sequence set forth in SEQ ID NO:15.

Provided is an isolated nucleic acid comprising a BAAV p19 promoter. Thenucleic acid can consist of the sequence set forth in SEQ ID NO:16. Thenucleic acid can consist essentially of the sequence set forth in SEQ IDNO:16. Further provided is a nucleic acid that selectively hybridizeswith the sequence set forth in SEQ ID NO:16.

Provided is an isolated nucleic acid comprising a BAAV p40 promoter. Thenucleic acid can consist of the sequence set forth in SEQ ID NO:17. Thenucleic acid can consist essentially of the sequence set forth in SEQ IDNO:17. Further provided is a nucleic acid that selectively hybridizeswith the sequence set forth in SEQ ID NO:17.

Provided is an isolated nucleic acid comprising a BAAV ITR. The isolatednucleic acid can comprise the sequence set forth in SEQ ID NO:12. Theisolated nucleic acid can consist essentially of the sequence set forthin SEQ ID NO:12. Further provided is an isolated nucleic acid thatselectively hybridizes with the sequence set forth in SEQ ID NO:12.

Further provided is an isolated nucleic acid encoding a bovineadeno-associated virus Rep protein. The BAAV Rep proteins are encoded byopen reading frame (ORF) 1 of the BAAV genome. Examples of the BAAV Repgenes are shown in the nucleic acid set forth in SEQ ID NO:1, andinclude nucleic acids consisting essentially of the nucleotide sequencesset forth in SEQ ID NOS:2 (rep78), 4 (rep52) and nucleic acidscomprising the nucleotide sequences set forth in SEQ ID NOS:2 and 4.However, it is contemplated that the Rep nucleic acid can include anyone, two, three, or four of the four Rep proteins, in any order, in sucha nucleic acid.

Furthermore, minor modifications are contemplated in the nucleic acid,such as silent mutations in the coding sequences, mutations that makeneutral or conservative changes in the encoded amino acid sequence, andmutations in regulatory regions that do not disrupt the expression ofthe gene. Examples of other minor modifications are known in the art.Further modifications can be made in the nucleic acid, such as todisrupt or alter expression of one or more of the Rep proteins in orderto, for example, determine the effect of such a disruption; such as tomutate one or more of the Rep proteins to determine the resultingeffect, etc. However, in general, a modified nucleic acid encoding a Repprotein will have at least about 85%, about 90%, about 93%, about 95%,about 98% or 100% homology to the Rep nucleic sequences described hereine.g., SEQ ID NOS: 2, and 4, and the Rep polypeptide encoded therein willhave overall about 93%, about 95%, about 98%, about 99% or 100% homologywith the amino acid sequence described herein, e.g., SEQ ID NOS:3 and 5.Percent homology is determined by the techniques described herein.

Provided herein is an isolated nucleic acid that selectively orspecifically hybridizes with a nucleic acid consisting essentially ofthe nucleotide sequence set forth in SEQ ID NOS:2 and 4, and an isolatednucleic acid that selectively hybridizes with a nucleic acid comprisingthe nucleotide sequence set forth in SEQ ID NOS:2 and 4. “Selectivelyhybridizing” and “stringency of hybridization” is defined elsewhereherein.

As described above, provided is the nucleic acid encoding a Rep 78protein and, in particular an isolated nucleic acid comprising thenucleotide sequence set forth in SEQ ID NO: 2, an isolated nucleic acidconsisting essentially of the nucleotide sequence set forth in SEQ IDNO: 2, and a nucleic acid encoding the bovine adeno-associated virusprotein having the amino acid sequence set forth in SEQ ID NO: 3. Alsoprovided is the nucleic acid encoding a Rep 52 protein, and inparticular an isolated nucleic acid comprising the nucleotide sequenceset forth in SEQ ID NO:4, an isolated nucleic acid consistingessentially of the nucleotide sequence set forth in SEQ ID NO:4, and anucleic acid encoding the bovine adeno-associated virus Rep 52 proteinhaving the amino acid sequence set forth in SEQ ID NO:5. As describedelsewhere herein, these nucleic acids can have minor modifications,including silent nucleotide substitutions, mutations causingconservative amino acid substitutions in the encoded proteins, andmutations in control regions that do not or minimally affect the encodedamino acid sequence.

Further provided is an isolated nucleic acid encoding a BAAV Capsidprotein. Furthermore, provided is a nucleic acid encoding each of thethree BAAV capsid proteins, VP1, VP2, and VP3. Thus, provided is anisolated nucleic acid encoding BAAV VP1, a nucleic acid encoding BAAVVP2, and an isolated nucleic acid encoding BAAV VP3. Thus, provided isan isolated nucleic acid encoding the amino acid sequence set forth inSEQ ID NO:7 (VP1); an isolated nucleic acid encoding the amino acidsequence set forth in SEQ ID NO:9 (VP2), and an isolated nucleic acidencoding the amino acid sequence set forth in SEQ ID NO:11 (VP3). Alsospecifically provided is an isolated nucleic acid comprising SEQ ID NO:6(VP1 gene); an isolated nucleic acid comprising SEQ ID NO:8 (VP2 gene);and an isolated nucleic acid comprising SEQ ID NO:10 (VP3 gene). Alsospecifically provided is an isolated nucleic acid consisting essentiallyof SEQ ID NO:6 (VP1 gene), an isolated nucleic acid consistingessentially of SEQ ID NO:8 (VP2 gene), and an isolated nucleic acidconsisting essentially of SEQ ID NO:10 (VP3 gene). Minor modificationsin the nucleotide sequences encoding the capsid, or coat, proteins arecontemplated, as described above for other BAAV nucleic acids. However,in general, a modified nucleic acid encoding a capsid protein will haveat least about 85%, about 90%, about 93%, about 95%, about 98% or 100%homology to the capsid nucleic sequences described herein e.g., SEQ IDNOS: 6, 8, and 10, and the capsid polypeptide encoded therein will haveoverall about 93%, about 95%, about 98%, about 99% or 100% homology withthe amino acid sequence described herein, e.g., SEQ ID NOS:7, 9, and 11.Isolated nucleic acids that selectively hybridize with the nucleic acidsof SEQ ID NOS:6, 8 or 10 under the conditions described above are alsoprovided.

Also provided is a cell containing one or more of the herein describednucleic acids, such as the BAAV genome, BAAV ORF1 and ORF2, each BAAVRep protein gene, or each BAAV capsid protein gene. Such a cell can beany desired cell and can be selected based upon the use intended. Forexample, cells can include bacterial cells, yeast cells, insect cells,human HeLa cells and simian Cos cells as well as other human andmammalian cells and cell lines. Primary cultures as well as establishedcultures and cell lines can be used. Nucleic acids provided herein canbe delivered into cells by any selected means, in particular dependingupon the target cells. Many delivery means are well-known in the art.For example, electroporation, calcium phosphate precipitation,microinjection, cationic or anionic liposomes, and liposomes incombination with a nuclear localization signal peptide for delivery tothe nucleus can be utilized, as is known in the art. Additionally, ifthe nucleic acids are in a viral particle, the cells can simply betransduced with the virion by standard means known in the art for AAVtransduction. Small amounts of the recombinant BAAV virus can be made toinfect cells and produce more of itself.

Provided herein are purified BAAV polypeptides. The term “polypeptide”as used herein refers to a polymer of amino acids and includesfull-length proteins and fragments thereof. Thus, “protein,”polypeptide,” and “peptide” are often used interchangeably herein.Substitutions can be selected by known parameters to be neutral (see,e.g., Robinson W E Jr, and Mitchell W M., AIDS 4:S151-S162 (1990)). Aswill be appreciated by those skilled in the art, also provided hereinare those polypeptides having slight variations in amino acid sequencesor other properties. Such variations may arise naturally as allelicvariations (e.g., due to genetic polymorphism) or may be produced byhuman intervention (e.g., by mutagenesis of cloned DNA sequences), suchas induced point, deletion, insertion and substitution mutants. Minorchanges in amino acid sequence are generally preferred, such asconservative amino acid replacements, small internal deletions orinsertions, and additions or deletions at the ends of the molecules.Substitutions may be designed based on, for example, the model ofDayhoff, et al. (in Atlas of Protein Sequence and Structure 1978, Nat'lBiomed. Res. Found., Washington, D.C.). These modifications can resultin changes in the amino acid sequence, provide silent mutations, modifya restriction site, or provide other specific mutations. The location ofany modifications to the polypeptide will often determine its impact onfunction. Particularly, alterations in regions non-essential to proteinfunction will be tolerated with fewer effects on function. Elsewhere inthe application regions of the BAAV proteins are described to provideguidance as to where substitutions, additions or deletions can be madeto minimize the likelihood of disturbing the function of the variant.

Protein variants and derivatives are well understood to those of skillin the art and in can involve amino acid sequence modifications. Forexample, amino acid sequence modifications typically fall into one ormore of three classes: substitutional, insertional or deletionalvariants. Insertions include amino and/or carboxyl terminal fusions aswell as intrasequence insertions of single or multiple amino acidresidues. Insertions ordinarily will be smaller insertions than those ofamino or carboxyl terminal fusions, for example, on the order of one tofour residues. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. Typically, no more thanabout from 2 to 6 residues are deleted at any one site within theprotein molecule. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTables 1 and 2 and are referred to as conservative substitutions.

TABLE 1 Amino Acid Abbreviations Amino Acid Abbreviations alanine Ala Aallosoleucine AIle arginine Arg R asparagine Asn N aspartic acid Asp Dcysteine Cys C glutamic acid Glu E glutamine Gln Q glycine Gly Ghistidine His H isolelucine Ile I leucine Leu L lysine Lys Kphenylalanine Phe F proline Pro P pyroglutamic acid pGlu serine Ser Sthreonine Thr T tyrosine Tyr Y tryptophan Trp W valine Val V

TABLE 2 Amino Acid Substitutions Original Residue Exemplary ConservativeSubstitutions, others are known in the art. Ala Ser Arg Lys; Gln AsnGln; His Asp Glu Cys Ser Gln Asn, Lys Glu Asp Gly Pro His Asn; Gln IleLeu; Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr SerThr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Substantial changes in function or immunological identity can resultfrom selecting substitutions that are less conservative than those inTable 2, i.e., selecting residues that differ more significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site or (c) the bulk of the side chain. The substitutionswhich in general are expected to produce the greatest changes in theprotein properties will be those in which (a) a hydrophilic residue,e.g. seryl or threonyl, is substituted for (or by) a hydrophobicresidue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) acysteine or proline is substituted for (or by) any other residue; (c) aresidue having an electropositive side chain, e.g., lysyl, arginyl, orhistidyl, is substituted for (or by) an electronegative residue, e.g.,glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Generally, a conservative substitution is a substitution of an aminoacid residue for another amino acid residue having similar biochemicalproperties. Typically, conservative substitutions have little to noimpact on the biological activity of a resulting polypeptide. In aparticular example, a conservative substitution is an amino acidsubstitution in a peptide that does not substantially affect thebiological function of the peptide. A peptide can include one or moreamino acid substitutions, for example 2-10 conservative substitutions,2-5 conservative substitutions, 4-9 conservative substitutions, such as2, 5 or 10 conservative substitutions.

For example, a conservative substitution in VP3 peptide (such as apeptide encoded by SEQ ID NO:9) does not substantially affect theability of VP3 peptide to confer the unique tropism of the BAAVparticle. A polypeptide can be produced to contain one or moreconservative substitutions by manipulating the nucleotide sequence thatencodes that polypeptide using, for example, standard procedures such assite-directed mutagenesis or PCR. Alternatively, a polypeptide can beproduced to contain one or more conservative substitutions by usingstandard peptide synthesis methods. An alanine scan can be used toidentify which amino acid residues in a protein can tolerate an aminoacid substitution. In one example, the biological activity of theprotein is not decreased by more than 25%, for example not more than20%, for example not more than 10%, when an alanine, or otherconservative amino acid (such as those listed below), is substituted forone or more native amino acids.

Examples of amino acids which can be substituted for an original aminoacid in a protein and which are regarded as conservative substitutionsinclude, but are not limited to: Ser for Ala; Lys for Arg; Gln or Hisfor Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro forGly; Asn or Gln for His; Leu or Val for Ile; Ile or Val for Leu; Arg orGln for Lys; Leu or Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser;Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile or Leu for Val.

Further information about conservative substitutions can be found in,among other locations in, Ben-Bassat et al., (J. Bacteriol. 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988) and in standard textbooks of genetics and molecular biology.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that there are numerous amino acid and peptide analogswhich can be incorporated into the disclosed compositions. For example,there are numerous D amino acids or amino acids which have a differentfunctional substituent then the amino acids shown in Table 1 and Table2. The opposite stereo isomers of naturally occurring peptides aredisclosed, as well as the stereo isomers of peptide analogs. These aminoacids can readily be incorporated into polypeptide chains by chargingtRNA molecules with the amino acid of choice and engineering geneticconstructs that utilize, for example, amber codons, to insert the analogamino acid into a peptide chain in a site specific way (Thorson et al.,Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion inBiotechnology, 3:348-354 (1992); Ibba, Biotechnology & GeneticEngineering Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Bio/technology, 12:678-682 (1994) all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble peptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH2NH—, —CH2S—, —CH2-CH2-,—CH═CH— (cis and trans), —COCH2-, —CH(OH)CH2-, and —CHH2SO— (These andothers can be found in Spatola, A. F. in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, Peptide Backbone Modifications (general review); Morley, TrendsPharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res14:177-185 (1979) (—CH2NH—, CH2CH2-); Spatola et al. Life Sci38:1243-1249 (1986) (—CHH2-S); Hann J. Chem. Soc Perkin Trans. I 307-314(1982) (—CH—CH—, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (—COCH2-); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH2-); Szelke et al. European Appln, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH2-); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH2-); and Hruby Life Sci 31:189-199 (1982)(—CH2-S—); each of which is incorporated herein by reference. Aparticularly preferred non-peptide linkage is —CH2NH—. It is understoodthat peptide analogs can have more than one atom between the bond atoms,such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhancedor desirable properties, such as, more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers.

D-amino acids can be used to generate more stable peptides, because Damino acids are not recognized by peptidases and such. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) canbe used to generate more stable peptides. Cysteine residues can be usedto cyclize or attach two or more peptides together. This can bebeneficial to constrain peptides into particular conformations. (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference).

A polypeptide provided herein can be readily obtained by any of severalmeans. For example, the polypeptide of interest can be synthesizedchemically by standard methods. Additionally, the coding regions of thegenes can be recombinantly expressed and the resulting polypeptideisolated by standard methods. Furthermore, an antibody specific for theresulting polypeptide can be raised by standard methods (see, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1988), and the protein can beisolated from a cell expressing the nucleic acid encoding thepolypeptide by selective hybridization with the antibody. This proteincan be purified to the extent desired by standard methods of proteinpurification (see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989).

An antigenic or immunoreactive fragment of the provided compositions andmethods is typically an amino acid sequence of at least about 5consecutive amino acids, and it can be derived from the BAAV polypeptideamino acid sequence. An antigenic BAAV fragment is any fragment uniqueto the BAAV protein, as described herein, against which a BAAV-specificantibody can be raised, by standard methods. Thus, the resultingantibody-antigen reaction should be specific for BAAV.

By “unique fragment thereof” is meant any smaller polypeptide fragmentencoded by a BAAV rep gene that is of sufficient length to be found onlyin the Rep polypeptide. Substitutions and modifications of the aminoacid sequence can be made as described herein and, further, can includeprotein processing modifications, such as glycosylation, to thepolypeptide. Typically, to be unique, a polypeptide fragment providedherein will be at least about 5 amino acids in length; however, uniquefragments can be 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ormore amino acids in length. A unique polypeptide will typically comprisesuch a unique fragment; however, a unique polypeptide can also bedetermined by its overall homology. A unique polypeptide can be 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids inlength. Uniqueness of a polypeptide fragment can readily be determinedby standard methods such as searches of computer databases of knownpeptide or nucleic acid sequences or by hybridization studies to thenucleic acid encoding the protein or to the protein itself, as known inthe art. The uniqueness of a polypeptide fragment can also be determinedimmunologically as well as functionally. Uniqueness can be simplydetermined in an amino acid-by-amino acid comparison of thepolypeptides.

Provided is an isolated BAAV Rep protein. A BAAV Rep polypeptide isencoded by ORF1 of BAAV. Also provided is each individual BAAV Repprotein. Provided is an isolated polypeptide, comprising BAAV Rep 52, ora unique fragment thereof. BAAV Rep 52 can have the amino acid sequenceset forth in SEQ ID NO:5. BAAV Rep 52 protein can be encoded by thenucleic acid sequence set forth in SEQ ID NO:2, or a unique fragmentthereof. Provided is an isolated polypeptide, comprising BAAV Rep 78, ora unique fragment thereof. BAAV Rep 78 can have the amino acid sequenceset forth in SEQ ID NO:3. BAAV Rep 78 protein can be encoded by thenucleic acid sequence set forth in SEQ ID NO:4, or a unique fragmentthereof.

Further provided is an isolated BAAV Capsid protein or a unique fragmentthereof. BAAV capsid protein is encoded by ORF 2 of BAAV. Furtherprovided are the individual BAAV capsid proteins, VP1, VP2 and VP3 orunique fragments thereof. Thus, provided is an isolated polypeptidehaving the amino acid sequence set forth in SEQ ID NO:7 (VP1). Furtherprovided is an isolated polypeptide consisting essentially of the aminoacid sequence set forth in SEQ ID NO:7. Additionally provided is anisolated polypeptide having the amino acid sequence set forth in SEQ IDNO:9 (VP2). Further provided is an isolated polypeptide consistingessentially of the amino acid sequence set forth in SEQ ID NO:9. Alsoprovided is an isolated polypeptide having the amino acid sequence setforth in SEQ ID NO:11 (VP3). Further provided is an isolated polypeptideconsisting essentially of the amino acid sequence set forth in SEQ IDNO:11.

By “unique fragment thereof” is meant any smaller polypeptide fragmentencoded by any BAAV capsid gene that is of sufficient length to be foundonly in the BAAV capsid protein. Substitutions and modifications of theamino acid sequence can be made as described above and, further, caninclude protein processing modifications, such as glycosylation, to thepolypeptide. However, a BAAV Capsid polypeptide including all three coatproteins will have greater than about 56% overall homology to thepolypeptide encoded by the nucleotides set forth in SEQ ID NOS:6, 8 or10. The protein can have about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, 93%, 95%, 97% or even 100% homology to the aminoacid sequence encoded by the nucleotides set forth in SEQ ID NOS:6, 8 or10. A BAAV VP1 polypeptide can have at least about 58%, about 60%, about70%, about 80%, about 90%, 93%, 95%, 97% or about 100% homology to theamino acid sequence set forth in SEQ ID NO:7. A BAAV VP2 polypeptide canhave at least about 58%, about 60%, about 70%, about 80%, about 90%,93%, 95%, 97% or about 100% homology to the amino acid sequence setforth in SEQ ID NO:9. A BAAV VP3 polypeptide can have at least about60%, about 70%, about 80%, about 90%, 93%, 95%, 97% or about 100%homology to the amino acid sequence set forth in SEQ ID NO:11.

Further provided is an isolated antibody that specifically binds a BAAVRep protein or a unique epitope thereof. Also provided are isolatedantibodies that specifically bind the BAAV Rep 52 protein and the BAAVRep 78 protein having the amino acid sequences set forth in SEQ ID NO: 5and SEQ ID NO: 3, respectively or that specifically binds a uniquefragment thereof. Clearly, any given antibody can recognize and bind oneof a number of possible epitopes present in the polypeptide; thus only aunique portion of a polypeptide (having the epitope) may need to bepresent in an assay to determine if the antibody specifically binds thepolypeptide.

Additionally provided is an isolated antibody that specifically bindsany of the bovine adeno-associated virus capsid proteins (VP1, VP2 orVP3), a unique epitope thereof, or the polypeptide comprising all threeBAAV coat proteins. Also provided is an isolated antibody thatspecifically binds the BAAV capsid protein having the amino acidsequence set forth in SEQ ID NO:7 (VP1), or that specifically binds aunique fragment thereof. Further provided is an isolated antibody thatspecifically binds the BAAV Capsid protein having the amino acidsequence set forth in SEQ ID NO:9 (VP2), or that specifically binds aunique fragment thereof. Additionally provided is an isolated antibodythat specifically binds the BAAV Capsid protein having the amino acidsequence set forth in SEQ ID NO:11 (VP3), or that specifically binds aunique fragment thereof. Again, any given antibody can recognize andbind one of a number of possible epitopes present in the polypeptide;thus only a unique portion of a polypeptide (having the epitope) mayneed to be present in an assay to determine if the antibody specificallybinds the polypeptide.

The antibody can be a component of a composition that comprises anantibody that specifically binds the BAAV protein. The composition canfurther comprise, e.g., serum, serum-free medium, or a pharmaceuticallyacceptable carrier such as physiological saline, etc.

By “an antibody that specifically binds” a BAAV polypeptide or proteinis meant an antibody that selectively binds to an epitope on any portionof the BAAV peptide such that the antibody binds specifically to thecorresponding BAAV polypeptide without significant background. Specificbinding by an antibody further means that the antibody can be used toselectively remove the target polypeptide from a sample comprising thepolypeptide or and can readily be determined by radioimmunoassay (RIA),bioassay, or enzyme-linked immunosorbant (ELISA) technology. An ELISAmethod effective for the detection of the specific antibody-antigenbinding can, for example, be as follows: (1) bind the antibody to asubstrate; (2) contact the bound antibody with a sample containing theantigen; (3) contact the above with a secondary antibody bound to adetectable moiety (e.g., horseradish peroxidase enzyme or alkalinephosphatase enzyme); (4) contact the above with the substrate for theenzyme; (5) contact the above with a color reagent; (6) observe thecolor change.

An antibody can include antibody fragments such as Fab fragments whichretain the binding activity. Antibodies can be made as described in,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1988). Briefly, purifiedantigen can be injected into an animal in an amount and in intervalssufficient to elicit an immune response. Antibodies can either bepurified directly, or spleen cells can be obtained from the animal. Thecells are then fused with an immortal cell line and screened forantibody secretion. Individual hybridomas are then propagated asindividual clones serving as a source for a particular monoclonalantibody.

Additionally provided is a method of screening a cell for infectivity byBAAV, comprising contacting the cell with BAAV and detecting thepresence of BAAV in the cells. BAAV particles can be detected using anystandard physical or biochemical methods. For example, physical methodsthat can be used for this detection include DNA based methods such as 1)polymerase chain reaction (PCR) for viral DNA or RNA or 2) directhybridization with labeled probes, and immunological methods such as by3) antibody directed against the viral structural or non-structuralproteins. Catalytic methods of viral detection include, but are notlimited to, detection of site and strand specific DNA nicking activityof Rep proteins or replication of an AAV origin-containing substrate.Reporter genes can also be utilized to detect cells that transduce BAAV.For example, β-gal, green fluorescent protein or luciferase can beinserted into a recombinant BAAV. The cell can then be contacted withthe recombinant BAAV, either in vitro or in vivo and a colorimetricassay could detect a color change in the cells that would indicatetransduction of BAAV in the cell. Additional detection methods areoutlined in Fields, Virology, Raven Press, New York, N.Y. 1996.

Provided is a method of screening a cell for infectivity by BAAV,wherein the presence of BAAV in the cells is determined by nucleic acidhybridization methods, a nucleic acid probe for such detection cancomprise, for example, a unique fragment of any of the BAAV nucleicacids provided herein. The uniqueness of any nucleic acid probe canreadily be determined as described herein. Additionally, the presence ofBAAV in cells can be determined by fluorescence, antibodies to geneproducts, focus forming assays, plaque lifts, Western blots andchromogenic assays. The nucleic acid can be, for example, the nucleicacid whose nucleotide sequence is set forth in SEQ ID NO: 1, 3, 4, 6, 8,10, 12, 13, 14, 15, 16, 17 or a unique fragment thereof.

Provided is a method of determining the suitability of a BAAV vector foradministration to a subject comprising contacting an antibody-containingsample from the subject with an antigenic fragment of an isolated BAAVRep or Capsid protein, and detecting an antibody-antigen reaction in thesample, the presence of a neutralizing reaction indicating the BAAVvector to be unsuitable for use in the subject. Further provided is amethod of determining the presence in a subject of a BAAV-specificantibody comprising contacting an antibody-containing sample from thesubject with an antigenic fragment of an isolated BAAV Rep or Capsidprotein and detecting an antibody-antigen reaction in the sample, thepresence of a reaction indicating the presence of a BAAV-specificantibody in the subject. The present methods of determining thesuitability of a BAAV vector for administration to a subject or thepresence of a BAAV-specific antibody in a subject can comprisecontacting an antibody-containing sample from the subject with a uniqueantigenic or immunogenic fragment of a BAAV Rep protein (e.g. Rep 52,Rep 78) and detecting an antibody-antigen reaction in the sample, thepresence of a reaction indicating the presence of a BAAV-specificantibody and therefore the BAAV vector to be unsuitable for use in thesubject. The BAAV Rep proteins are provided herein, and their antigenicfragments are routinely determined. The BAAV capsid protein can be usedto select an antigenic or immunogenic fragment, for example from theamino acid sequence set forth in SEQ ID NO:7 (VP1), the amino acidsequence set forth in SEQ ID NO: 9 (VP2) or the amino acid sequence setforth in SEQ ID NO:11 (VP3). Alternatively, or additionally, anantigenic or immunogenic fragment of an isolated BAAV Rep protein can beutilized in this determination method. The BAAV Rep protein from whichan antigenic fragment is selected can have the amino acid sequenceencoded by the nucleic acid set forth in SEQ ID NO:1, the amino acidsequence set forth in SEQ ID NO:2, or the amino acid sequence set forthin SEQ ID NO:4, the amino acid sequence set forth in SEQ ID NO: 3, orthe amino acid sequence set forth in SEQ ID NO:5.

The BAAV polypeptide fragments can be analyzed to determine theirantigenicity, immunogenicity and/or specificity. Briefly, variousconcentrations of a putative immunogenically specific fragment areprepared and administered to a subject and the immunological response(e.g., the production of antibodies or cell mediated immunity) of ananimal to each concentration is determined. The amounts of antigenadministered depend on the subject, e.g. a human, rabbit or a guineapig, the condition of the subject, the size of the subject, etc.Thereafter an animal so inoculated with the antigen can be exposed tothe BAAV viral particle or BAAV protein to test the immunoreactivity orthe antigenicity of the specific immunogenic fragment. The specificityof a putative antigenic or immunogenic fragment can be ascertained bytesting sera, other fluids or lymphocytes from the inoculated animal forcross reactivity with other closely related viruses, such as AAV1-8 orAAAV.

By the “suitability of a BAAV vector for administration to a subject” ismeant a determination of whether the BAAV vector will elicit aneutralizing immune response upon administration to a particularsubject. A vector that does not elicit a significant immune response isa potentially suitable vector, whereas a vector that elicits asignificant, neutralizing immune response (e.g. at least 90%) is thuslikely to be unsuitable for use in that subject. Significance of anydetectable immune response is a standard parameter understood by theskilled artisan in the field. For example, one can incubate thesubject's serum with the virus, then determine whether that virusretains its ability to transduce cells in culture. If such virus cannottransduce cells in culture, the vector likely has elicited a significantimmune response.

Alternatively, or additionally, one skilled in the art could determinewhether or not BAAV administration would be suitable for a particularcell type of a subject. For example, the artisan could culture musclecells in vitro and transduce the cells with BAAV in the presence orabsence of the subject's serum. If there is a reduction in transductionefficiency, this could indicate the presence of a neutralizing antibodyor other factors that may inhibit transduction. Normally, greater than90% inhibition would have to be observed in order to rule out the use ofBAAV as a vector. However, this limitation could be overcome by treatingthe subject with an immunosuppressant that could block the factorsinhibiting transduction.

As will be recognized by those skilled in the art, numerous types ofimmunoassays are available for use in the present methods to detectbinding between an antibody and a BAAV polypeptide as provided herein.For instance, direct and indirect binding assays, competitive assays,sandwich assays, and the like, as are generally described in, e.g., U.S.Pat. Nos. 4,642,285; 4,376,110; 4,016,043; 3,879,262; 3,852,157;3,850,752; 3,839,153; 3,791,932; and Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, N.Y. (1988). Forexample, enzyme immunoassays such as immunofluorescence assays (IFA),enzyme linked immunosorbent assays (ELISA) and immunoblotting can bereadily adapted to accomplish the detection of the antibody. An ELISAmethod effective for the detection of the antibody bound to the antigencan, for example, be as follows: (1) bind the antigen to a substrate;(2) contact the bound antigen with a fluid or tissue sample containingthe antibody; (3) contact the above with a secondary antibody specificfor the antigen and bound to a detectable moiety (e.g., horseradishperoxidase enzyme or alkaline phosphatase enzyme); (4) contact the abovewith the substrate for the enzyme; (5) contact the above with a colorreagent; (6) observe color change.

The antibody-containing sample of this method can comprise anybiological sample which would contain the antibody or a cell containingthe antibody, such as blood, plasma, serum, bone marrow, saliva andurine.

Also provided is a method of producing the BAAV virus by transducing acell with the nucleic acid encoding the virus.

The present method further provides a method of delivering an exogenousnucleic acid to a cell comprising administering to the cell a BAAVparticle containing a vector comprising the nucleic acid insertedbetween a pair of AAV inverted terminal repeats, thereby delivering thenucleic acid to the cell.

The AAV ITRs in the vector for the herein described delivery methods canbe BAAV ITRs (SEQ ID NOS: 12). Furthermore, the AAV ITRs in the vectorfor the herein described nucleic acid delivery methods can also compriseAAV1, 2, 3, 4, 5, 6, 7, 8 or AAAV inverted terminal repeats.

Also provided is a method of delivering an exogenous nucleic acid to asubject comprising administering to a cell of or from the subject a BAAVparticle containing a vector comprising the nucleic acid insertedbetween a pair of AAV inverted terminal repeats, and returning the cellto the subject, thereby delivering the nucleic acid to the subject. TheAAV ITRs can be any AAV ITRs, including BAAV ITRs, AAV5 ITRs and AAV2ITRs. For example, in an ex vivo administration, cells are isolated froma subject by standard means according to the cell type and placed inappropriate culture medium, again according to cell type (see, e.g.,ATCC catalog). Viral particles are then contacted with the cells asdescribed above, and the virus is allowed to transduce the cells. Cellscan then be transplanted back into the subject's body, again by meansstandard for the cell type and tissue (e.g., in general, U.S. Pat. No.5,399,346; for neural cells, Dunnett, S. B. and Björklund, A., eds.,Transplantation: Neural Transplantation—A Practical Approach, OxfordUniversity Press, Oxford (1992)). If desired, prior to transplantation,the cells can be studied for degree of transduction by the virus, byknown detection means and as described herein. Cells for ex vivotransduction followed by transplantation into a subject can be selectedfrom those listed above, or can be any other selected cell. Preferably,a selected cell type is examined for its capability to be transfected byBAAV. Preferably, the selected cell will be a cell readily transducedwith BAAV particles; however, depending upon the application, even cellswith relatively low transduction efficiencies can be useful,particularly if the cell is from a tissue or organ in which evenproduction of a small amount of the protein or antisense RNA encoded bythe vector will be beneficial to the subject.

Further provided is a method of delivering an exogenous nucleic acid toa cell in a subject comprising administering to the subject a BAAVparticle containing a vector comprising the nucleic acid insertedbetween a pair of AAV inverted terminal repeats, thereby delivering thenucleic acid to a cell in the subject. Administration can be an ex vivoadministration directly to a cell removed from a subject, such as any ofthe cells listed above, followed by replacement of the cell back intothe subject, or administration can be in vivo administration to a cellin the subject. For ex vivo administration, cells are isolated from asubject by standard means according to the cell type and placed inappropriate culture medium, again according to cell type (see, e.g.,ATCC catalog). Viral particles are then contacted with the cells asdescribed above, and the virus is allowed to transfect the cells. Cellscan then be transplanted back into the subject's body, again by meansstandard for the cell type and tissue (e.g., for neural cells, Dunnett,S. B. and Björklund, A., eds., Transplantation: Neural Transplantation—APractical Approach, Oxford University Press, Oxford (1992)). If desired,prior to transplantation, the cells can be studied for degree oftransfection by the virus, by known detection means and as describedherein.

Further provided is a method of delivering a nucleic acid to a cell in asubject having neutralizing antibodies to AAV1-8 comprisingadministering to the subject a BAAV particle containing a vectorcomprising the nucleic acid, thereby delivering the nucleic acid to acell in the subject. A subject that has neutralizing antibodies toAAV1-8 can readily be determined by any of several known means, such ascontacting AAV1-8 protein(s) with an antibody-containing sample, such asblood, from a subject and detecting an antigen-antibody reaction in thesample. Delivery of the AAV1-8 particle can be by either ex vivo or invivo administration as herein described. Thus, a subject who might havean adverse immunogenic reaction to a vector administered in an AAV2viral particle can have a desired nucleic acid delivered using an AAV1-8particle. This delivery system can be particularly useful for subjectswho have received therapy utilizing AAV1-8 particles in the past andhave developed antibodies to AAV1-8. A BAAV regimen can now besubstituted to deliver the desired nucleic acid.

In any of the methods of delivering exogenous nucleic acids to a cell orsubject described herein, the BAAV-conjugated nucleic acid or BAAVparticle-conjugated nucleic acids described herein can be used.

In vivo administration to a human subject or an animal model can be byany of many standard means for administering viruses, depending upon thetarget organ, tissue or cell. Virus particles can be administeredorally, parenterally (e.g., intravenously), by intramuscular injection,intrarectally, by direct tissue or organ injection, by intraperitonealinjection, topically, transdermally, via aerosol delivery, via themucosa or the like. Viral nucleic acids (non-encapsidated) can also beadministered, e.g., as a complex with cationic liposomes, orencapsulated in anionic liposomes. The present compositions can includevarious amounts of the selected viral particle or non-encapsidated viralnucleic acid in combination with a pharmaceutically acceptable carrierand, in addition, if desired, may include other medicinal agents,pharmaceutical agents, carriers, adjuvants, diluents, etc. Parentaladministration, if used, is generally characterized by injection.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Dosages willdepend upon the mode of administration, the disease or condition to betreated, and the individual subject's condition, but will be that dosagetypical for and used in administration of other AAV vectors, such asAAV2 vectors. Often a single dose can be sufficient; however, the dosecan be repeated if desirable. Administration methods for gene deliveryto the cochlea are routine and are described in Jero, J. et al. (GeneTher. 2001 Mar. 20; 12(5):539-48) and Staecker H, et al. (ActaOtolaryngol. 2001 January; 121(2):157-63), both references hereinincorporated by reference for these methods.

Administration methods can be used to treat brain disorders such asParkinson's disease, Alzheimer's disease, and demyelination disease.Other diseases that can be treated by these methods include metabolicdisorders such as musculoskeletal diseases, cardiovascular disease,cancer, and autoimmune disorders.

Administration of this recombinant BAAV virion to the cell can beaccomplished by any means, including simply contacting the particle,optionally contained in a desired liquid such as tissue culture medium,or a buffered saline solution, with the cells. The virion can be allowedto remain in contact with the cells for any desired length of time, andtypically the virion is administered and allowed to remain indefinitely.For such in vitro methods, the virion can be administered to the cell bystandard viral transduction methods, as known in the art and asexemplified herein. Titers of virus to administer can vary, particularlydepending upon the cell type, but will be typical of that used for AAVtransduction in general which is well known in the art. Additionally thetiters used to transduce the particular cells in the present examplescan be utilized.

The cells that can be transduced by the present recombinant BAAV virioncan include any desired cell, such as the following cells and cellsderived from the following tissues, human as well as other mammaliantissues, such as primate, horse, sheep, goat, pig, dog, rat, and mouseand avian species: Adipocytes, Adenocyte, Adrenal cortex, Amnion, Aorta,Ascites, Astrocyte, Bladder, Bone, Bone marrow, Brain, Breast, Bronchus,Cardiac muscle, Cecum, Cervix, Chorion, Cochlear, Colon, Conjunctiva,Connective tissue, Cornea, Dermis, Duodenum, Embryonic stem cells,Endometrium, Endothelium, Endothelial cells, Epithelial tissue,Epithelial cells, Epidermis, Esophagus, Eye, Fascia, Fibroblasts,Foreskin, Gastric, Glial cells, Glioblast, Gonad, Hepatic cells,Histocyte, Hair cells in the inner ear, auditory (organ of Corti)sensory epithelia, vestibular sensory epithelia, Ileum, Intestine, smallIntestine, Jejunum, Keratinocytes, Kidney, Larynx, Leukocytes, Lipocyte,Liver, Lung, Lymph node, Lymphoblast, Lymphocytes, Macrophages, Mammaryalveolar nodule, Mammary gland, Mastocyte, Maxilla, Melanocytes,Mesenchymal, Monocytes, Mouth, Myelin, Myoblasts Nervous tissue,Neuroblast, Neurons, Neuroglia, Osteoblasts, Osteogenic cells, Ovary,Palate, Pancreas, Papilloma, Peritoneum, Pituicytes, Pharynx, Placenta,Plasma cells, Pleura, Prostate, Rectum, Salivary gland, Skeletal muscle,Skin, Smooth muscle, Somatic, Spleen, Squamous, Stem cells, Stomach,Submandibular gland, Submaxillary gland, Synoviocytes, Testis, Thymus,Thyroid, Trabeculae, Trachea, Turbinate, Umbilical cord, Ureter, Uterus,and vestibular hair cells.

The cell of the provided methods can be an inner ear epithelial cell.Thus, the cell of the provided method can be an inner ear hair cell. Thecell of the provided methods can be an inner or outer hair cell of theorgan of Corti or a vestibular hair cell. The cell of the providedmethods can be an inner ear supporting cell such as Hensen's, phalangal,interdental, or vestibular supporting cells.

The cell of the provided method can be an airway epithelial cell. Thecell of the provided method can be a columnar, goblet or basal cell.

The cell of the provided method can be a cell of the submandibulargland. The cell of the provided method can be a ductal or acinar cell.

Provided are recombinant vectors based on BAAV. Such vectors may beuseful for transducing erythroid progenitor cells or cells resistant totransduction by other serotypes of AAV. These vectors may also be usefulfor transducing cells with a nucleic acid of interest in order toproduce cell lines that could be used to screen for agents that interactwith the gene product of the nucleic acid of interest. In addition totransduction of other cell types, transduction of erythroid cells wouldbe useful for the treatment of cancer and genetic diseases which can becorrected by bone marrow transplants using matched donors. Some examplesof this type of treatment include, but are not limited to, theintroduction of a therapeutic gene such as genes encoding interferons,interleukins, tumor necrosis factors, adenosine deaminase, cellulargrowth factors such as lymphokines, blood coagulation factors such asfactor VIII and IX, cholesterol metabolism uptake and transport proteinsuch as EpoE and LDL receptor, and antisense sequences to inhibit viralreplication of, for example, hepatitis or HIV.

Provided is a vector, comprising the BAAV virus as well as BAAV viralparticles. While BAAV is similar to AAV1-8, the viruses are found hereinto be physically and genetically distinct. These differences endow BAAVwith some unique advantages, which better suit it as a vector for genetherapy.

Furthermore, as shown herein, BAAV capsid protein is distinct fromAAV1-8 and AAAV capsid protein and exhibits different tissue tropism.AAV 1-8 and BAAV likely utilize distinct cellular receptors. AAV 1-8 andBAAV are serologically distinct and humans are not reported to haveneutralizing antibodies to BAAV, thus in a gene therapy or gene transferapplication, BAAV would allow for transduction of a patient who alreadypossess neutralizing antibodies to AAV1-8 either as a result of naturalimmunological defense or from prior exposure to AAV1-8 vectors.

Vector System

Provided herein is a vector system for producing infectious virusparticles having a characteristic of BAAV. As used herein, a “vectorsystem” is a combination of one or more vectors that, when added to anappropriate cell system, can produce a recombinant BAAV virion, asprovided herein.

The provided vector system can comprise: at least one vector comprisinga nucleic acid selected from the group consisting of a pair of BAAVITRs, a nucleic acid encoding a BAAV capsid protein, and a nucleic acidencoding a BAAV Rep protein.

The vector system can comprise one or more unique vectors. Thus, thevector system can comprise, for example, 1, 2, 3, 4, 5, or 6 uniquevectors.

In a two-vector vector system, the first vector can comprise a nucleicacid encoding a BAAV Rep protein and the second vector can comprise apair of BAAV ITRs. Alternatively, the first vector can comprise anucleic acid encoding a BAAV capsid protein and a nucleic acid encodinga BAAV Rep protein and the second vector can comprise a pair of BAAVITRs.

In another two-vector vector system, the first vector can comprise anucleic acid encoding a BAAV capsid protein and the second vector cancomprise a pair of AAV ITRs. The AAV ITRs of the second vector can be apair of AAV1 ITRs. The AAV inverted terminal repeats can be a pair ofAAV2 ITRs. The AAV ITRs can be a pair of AAV3 ITRs. The AAV ITRs can bea pair of AAV4 ITRs. The AAV ITRs can be a pair of AAV5 ITRs. The AAVITRs can be a pair of AAV6 ITRs. The AAV ITRs can be a pair of AAV7ITRs. The AAV ITRs can be a pair of AAV8 ITRs. The AAV ITRs can be apair of AAAV ITRs. The AAV ITRs can be a pair of BAAV ITRs.

The first vector can further comprise a nucleic acid encoding an AAV Repprotein. The AAV Rep protein can be AAV1 Rep protein. The AAV Repprotein can be AAV2 Rep protein. The AAV Rep protein can be AAV3 Repprotein. The AAV Rep protein can be AAV4 Rep protein. The AAV Repprotein can be AAV5 Rep protein. The AAV Rep protein can be AAV6 Repprotein. The AAV Rep protein can be AAV7 Rep protein. The AAV Repprotein can be AAV8 Rep protein. The AAV Rep protein can be AAAV Repprotein. The AAV Rep protein can be BAAV Rep protein. The Rep proteinscan be encoded by the nucleic acid sequence SEQ ID NOS:______.

In another two-vector system, the first vector can comprise a nucleicacid encoding an AAV capsid protein and the second vector can comprise apair of BAAV ITRs. The capsid protein can be an AAV1 capsid protein. Thecapsid protein can be an AAV2 capsid protein. The capsid protein can bean AAV3 capsid protein. The capsid protein can be an AAV4 capsidprotein. The capsid protein can be an AAV5 capsid protein. The capsidprotein can be an AAV6 capsid protein. The capsid protein can be a AAAVRep protein. The capsid protein can be a BAAV Rep protein.

The second vector can further comprise a promoter between the ITRs. Thepromoter can be AAV2 p5 promoter. The promoter can be AAV5 p5 promoter.The promoter can be BAAV p5 promoter. More specifically, the BAAV p5promoter can be in about the same location in SEQ ID NO: 1 as the AAV2p5 promoter, in the corresponding AAV2 published sequence. Additionally,the p5 promoter may be enhanced by nucleotides 1-173 of SEQ ID NO:1.Furthermore, smaller fragments of p5 promoter that retain promoteractivity can readily be determined by standard procedures including, forexample, constructing a series of deletions in the p5 promoter, linkingthe deletion to a reporter gene, and determining whether the reportergene is expressed, i.e., transcribed and/or translated. The promoter canbe the BAAV p19 promoter (SEQ ID NO: 16). The promoter can be the BAAVp40 promoter (SEQ ID NO: 17). The promoter can be a promoter of any ofthe AAV serotypes. The promoter can be a constitutive promoter. Thus,the promoter can be CMV. The promoter can be RSV. The promoter can beLTR. The promoter can be eF1. The promoter can be beta actin promoter.The promoter can be a tissue specific promoter. The promoter can be aninducible promoter. The promoter can further be functionally linked toan exogenous nucleic acid.

Further provided is any of the disclosed vectors of the vector systemsencapsidated into an AAV particle. The AAV particle can be an AAV1 virusparticle comprising at least one AAV1 capsid protein. The AAV particlecan be an AAV2 virus particle comprising at least one AAV2 capsidprotein. The AAV particle can be an AAV3 virus particle comprising atleast one AAV3 capsid protein. The AAV particle can be an AAV4 virusparticle comprising at least one AAV4 capsid protein. The AAV particlecan be an AAV5 virus particle comprising at least one AAV5 capsidprotein. The AAV particle can be an AAV6 virus particle comprising atleast one AAV6 capsid protein. The AAV particle can be an AAV7 virusparticle comprising at least one AAV7 capsid protein. The AAV particlecan be an AAV8 virus particle comprising at least one AAV8 capsidprotein. The AAV particle can be an AAAV virus particle comprising atleast one AAAV capsid protein. The AAV particle can be a BAAV virusparticle comprising at least one BAAV capsid protein. The AAV particlecan be a chimeric capsid virus particle (described above) comprising acapsid protein from more than one serotype of AAV.

AAV Transcytosis

Disclosed is a method of delivering an exogenous nucleic acid across anepithelial barrier, comprising delivering to the epithelial barrier anAAV vector, comprising the exogenous nucleic acid. In one aspect of themethod, the AAV is AAV4, AAV5, or BAAV. In another aspect of the method,the epithelial cells are in the gut, lung, genitourinary tract, kidney,blood vessels or brain. In another aspect of the method, the epithelialcells can be selected from a group consisting of bronchial, alveolar,tracheal or upper airway epithelial cells; absorptive enterocytes or Mcells; endometrial or urinary epithelial cells; renal collecting duct orproximal tubule epithelial cells; cerebral microvascular endothelialcells or Choroidal Plexus epithelial cells.

Further disclosed is a method of transcytosing epithelial cells of ahuman subject, comprising administering to the subject an AAV vectorcomprising an exogenous nucleic acid. In one aspect of the method, thevector is AAV4, AAV5, or BAAV. In another aspect of the method, theepithelial cells are selected from a group consisting of bronchial,alveolar, tracheal or upper airway epithelial cells; absorptiveenterocytes or M cells; endometrial or urinary epithelial cells; renalcollecting duct or proximal tubule epithelial cells; cerebralmicrovascular endothelial cells or Choroidal Plexus epithelial cells.

Further contemplated are methods for the delivery of molecules acrossepithelial cell barriers comprising coupling the molecules tonon-recombinant (wild-type) AAV capsids or particles. In one aspect, themolecules are radioligands or enzymes.

The term “adeno-associated virus (AAV)” is used herein to refer to agenus of viruses in the family Parvoviridae which are all defectiveviruses (unable to replicate by themselves) and depend on theco-infection of their host cell by other, nondefective viruses to helpthem replicate.

The term “transcytosis” is used herein to mean the transport ofmacromolecular cargo from one side of a cell to the other within amembrane-bounded carrier(s). Tuma and Hubbard provided a review oftranscytosis (Tuma P L and Hubbard A L. 2003. Physiol Rev. 83:871-932),herein incorporated by reference for its teaching regarding the natureand uses for trancytosis. Transcytosis is a strategy used bymulticellular organisms to selectively move material between twodifferent environments while maintaining the distinct compositions ofthose environments. N. Simionescu was the first to coin the termtranscytosis to describe the vectorial transfer of macromolecular cargowithin the plasmalemmal vesicles from the circulation across capillaryendothelial cells to the interstitium of tissues. During this sameperiod, another type of transcytosis was being discovered. Immunologistscomparing the different types of immunoglobulins found in varioussecretions (e.g., serum, milk, saliva, and the intestinal lumen)speculated that the form of IgA found in external secretions (calledsecretory IgA, due to the presence of an additional protein component)was selectively transported across the epithelial cell barrier. More isknown about transcytosis as it is expressed in epithelial tissues, whichform cellular barriers between two environments. In this polarized celltype, net movement of material can be in either direction, apical tobasolateral or the reverse, depending on the cargo and particularcellular context of the process. However, transcytosis is not restrictedto only epithelial cells.

Since the 19th century dye experiments of Ehrlich, the brain has beenknown as a “privileged” organ where access is tightly regulated so thatthe environment remains chemically stable. The two principal gatekeepersof the brain are the cerebral capillary endothelium and the cuboidalepithelial cells of the choroid plexus. These cellular barriers arespecialized for the passage of different nutrients from the blood. Thecapillaries move nutrients that are required rapidly and in largequantities, such as glucose and amino acids. These small molecules aretransported by membrane carriers using facilitated diffusion. Thechoroid plexus supplies nutrients that are required less acutely and inlower quantities. These are folate and other vitamins, ascorbate, anddeoxyribonucleotides.

There are two epithelial cells that participate in transcytosis in theintestine, M cells and enterocytes (adsorptive columnar cells). Thesecells are very different from one another and the capillary endothelialcell. Depending on the species, M cells comprise a variable but smallpercentage of the epithelia overlying organized mucosal-associatedlymphoid tissue, making them a very minor cell population in thegastrointestinal tract. The transcytotic route across M cells is thoughtto be part of the mechanism by which antigens are routinely sampledalong the entire mucosal surface. Not surprisingly, numerous pathogenshave evolved mechanisms to exploit the transcytotic process as a meansto invade and disseminate before a strong enough immune response can bemounted.

Absorptive enterocytes are simple columnar cells with several apicalfeatures in addition to their brush borders. Clathrin-coated pits arepresent at the base of microvilli, and a thick glycocalyx composed ofintegral membrane proteins with glycosaminoglycan side chains emanatesfrom the microvillar membrane. This latter structural feature as well asthe rigidity of the microvilli are thought to prohibit microorganismsfrom attaching and invading enterocytes. The intracellular organizationof these columnar epithelial cells is also polarized, with basallylocated nuclei, supranuclear Golgi, and an abundance of pleiomorphicmembrane compartments underlying the terminal web of the brush border.The basolateral-to-apical length of this cell is ˜20 versus 0.2 μm for acapillary endothelial cell, making the transcytotic route acrossenterocytes potentially much longer. Furthermore, microtubules are animportant structural element of the transcytotic pathway in enterocytes,but not in M or endothelial cells.

Transcytosis also occurs in the upper regions of the respiratory tractand has been demonstrated with two vector systems, pIgA-R and FcRn, butothers could exist. Secretory IgA is a known constituent of the lung'simmune defense system, with bronchial epithelial cells carrying outbasolateral-to-apical transport of dIgA, which is secreted by localplasma cells in underlying lymphoid tissue. Albumin, which is found inlung fluid, is endocytosed specifically at the apical surface of airwayepithelia but is then subsequently degraded. At the alveolar level, thequestion of whether albumin is transcytosed intact is uncertain.

The term “epithelia” is used herein to refer to cells which are linkedtightly together by intercellular junctions to form a planar sheet.These sheets of cells form a barrier between two compartments. Epitheliatherefore line all surfaces and cavities (including skin, peritoneum,linings of the intestine, airways, genitourinary tracts, glands, andblood vessels.

An epithelium has a free or apical surface facing the environment, orlumen of a cavity, and a basal surface facing the underlying connectivetissue. The boundary between the basal surface of an epithelium and theunderlying connective tissue is usually very sharp, and is the sitewhere the basal lamina (BL) is present. Most BL are too thin to be seenwith the light microscope. However, the BL, together with a thin layerof connective tissue, is often times seen at the epithelial/connectivetissue interface. This composite layer, visible with the lightmicroscope, was initially called the Basement Membrane. Application ofthe electron microscope revealed that, in most cases, this BasementMembrane actually consisted of the true basal lamina (lamina lucida pluslamina densa), along with a layer of adherent connective tissue.

For convenience of description, epithelia are classified into differenttypes based on the number of cell layers and the cell shape.

Epithelia which are 1 cell layer thick are called “simple” epithelia.Thus, each cell rests on the basal lamina, but also has a surface facingthe lumen/outside world. Epithelia which are 2 or more cell layers thickare called “stratified” epithelia. In stratified epithelia, the basallayer of cells rests on the basal lamina, but subsequent layers do not,and are simply stacked on top of the basal layer. The cells of the mostsuperficial layer have a free surface. “squamous” cells are very flat,like a fried egg, where the yolk is the nucleus. The nucleus isdistinctly flattened, the cell is often so thin that this flattenednucleus bulges the cell surface outward. “cuboidal” cells range fromtrue cuboidal where the cell is about as high as it is wide, to aflattened cuboidal where the cell is wider than high. In cuboidal cellsthe nucleus is usually round, and not flattened as in squamous.“columnar” cells are 2 or more times as high as wide. Nucleus is usuallyelongated in the long axis of the cell.

Squamous cells form the lining of cavities such as the mouth, bloodvessels, heart and lungs and make up the outer layers of the skin.Cuboidal epithelium is found in glands and in the lining of the kidneytubules as well as in the ducts of the glands. They also constitute thegerminal epithelium which produces the egg cells in the female ovary andthe sperm cells in the male testes. Columnar epithelium forms the liningof the stomach and intestines. Some columnar cells are specialized forsensory reception such as in the nose, ears and the taste buds of thetongue.

Ciliated columnar epithelial cells posses fine hair-like outgrowths,cilia on their free surfaces. These cilia are capable of rapid,rhythmic, wavelike beatings in a certain direction. Ciliated epitheliumis usually found in the air passages like the nose. It is also found inthe uterus and Fallopian tubes of females.

Columnar epithelium with goblet cells is called glandular epithelium.Some parts of the glandular epithelium consist of such a large number ofgoblet cells that there are only a few normal epithelial cells left.Columnar and cuboidal epithelial cells often become specialized as glandcells which are capable of synthesizing and secreting certain substancessuch as enzymes, hormones, milk, mucus, sweat, wax and saliva.Unicellular glands consist of single, isolated glandular cells such asthe goblet cells. Sometimes a portion of the epithelial tissue becomesinvaginated and a multicellular gland is formed. Multicellular glandsare composed of clusters of cells. Most glands are multicellularincluding the salivary glands.

Where body linings have to withstand wear and tear, the epithelia arecomposed of several layers of cells and are then called compound orstratified epithelium. The top cells are flat and scaly and it may ormay not be keratinized (i.e. containing a tough, resistant proteincalled keratin). The mammalian skin is an example of dry, keratinized,stratified epithelium. The lining of the mouth cavity is an example ofan unkeratinized, stratified epithelium.

The use of in vitro cell models to study transcytosis has manyadvantages over in vivo systems. First, variation among animals iseliminated, as is the confounding issue of cargo possibly being modifiedor endocytosed by cell types other than the one under study. Moreover,in vitro systems can be manipulated in ways not possible in vivo,allowing investigators to measure the effects of different variables(e.g., temperatures, pharmacological agents, etc.) with greaterprecision and to explore the molecular mechanisms of transcytosis.

The integrity of the monolayer is obviously vital to every study oftranscytosis, and there are different methods for assessing it.Transepithelial electrical resistance (TER) measurements are commonlyused as an indication of tight junction integrity in a monolayer, andcommercial instruments are available for these measurements.

Caco-2 cells, human primary colon carcinoma cells, are a well studiedmodel of intestinal absorptive enterocytes. They are the most commonlyused intestinal cell line because they differentiate furthest along thecryptto-villus axis and are the easiest to transfect. Caco-2 cells havebeen especially used to model transcytosis of bacteria, which can crossbarrier epithelia in the gut and brain (Zhang J R, et al., 2000. Cell102(6):827-37), incorporated herein by reference.

There is little evidence for in vivo transcytosis of macromolecularcargo in kidney. Nonetheless, MDCK cells, which are derived from dogkidney, are the most-studied epithelial cell model and have been usedextensively to study transcytosis. These cells were originally developedby nephrologists for permeability and electrical studies. Theirsubsequent use by cell biologists for studies of the formation of tightjunctions, establishment of polarity, and vesicle traffic havepopularized MDCK cells. An advantage is that MDCK cells are easilycultured, easily transfected, and become polarized 3-5 days afterseeding. They were used in the now classical studies showing thatenveloped viruses bud in a polarized fashion and that the newlysynthesized viral membrane glycoproteins are targeted directly from theTGN to the appropriate PM domain. Furthermore, much of the currentunderstanding of the IgA transcytotic pathway and the sorting signals inthe pIgA-R comes from the elegant studies performed in MDCK cells. TwoMDCK strains with very different features were identified some time ago.The MDCK I cell has a high TER and characteristics reminiscent of therenal collecting duct, whereas the more commonly used MDCK II strain,whose TER is one order of magnitude lower than that of MDCK I cells, hasphenotypic features closer to those of the renal proximal tubule.

Both primary cells and cell lines, alone and in coculture withendothelial cells, are being used to study transcytosis in the lung.Clonetics bronchial/tracheal epithelial cell systems contain normalhuman bronchial/treacheal epithelial cells. This cell system has beenused for experimental applications in cancer research, respiratorydisease, cellular function and differentiation.

The Clonetics® bovine Brain Microvascular Endothelial Cell System(bMVEC-B) is a model of the “Blood Brain Barrier”. The system isdesigned to significantly improve a researcher's ability to study activeand passive transport of drugs across the blood brain barrier, to studybrain endothelial cell tight junctions, and to study the basic biologyof brain microvascular endothelial cells (Schinket A H, 1999. AdvancedDrug Delivery Reviews 36:179-194; Tsukita S. et al., 1998. Moleculardissection of tight junctions: occluding and ZO-1 in Introduction to theBlood-Brain Barrier. Edited by William M Partridge; Inglis et al., 2004.Brain Research 998: 218-229), each of which is incorporated by referencefor its teaching of in vitro endothelial cell modeling of theblood-brain barrier.

Endometrial cells form an important barrier layer in the genitourinarytract. The cells used to model this system were developed by Kyo et al.and are derived from primary cells immortalized by the addition of thepapillomiavirus E6/E7 genes and human telomerase reverse transcriptase.The isolated cells have a normal chromosomes and retain theirresponsiveness to sex-steroid hormones, exhibit glandular structure onthree dimensional culture, and lack a transformed phenotype (Kyo S, etal. Am J Pathol., 2003. 163(6):2259-69), incorporated herein byreference for its teaching of this endometrial model.

The provided BAAV particles and virions combine the known advantages ofAAVs as vectors with distinct tropisms unique to BAAV viral particles. Afurther advantage of the provided compositions and methods is theability of BAAV virions to deliver nucleic acids across epithelialbarriers. Thus, the compositions and methods provided herein can be usedto deliver nucleic acids to cells or cells in a subject. The providedcompositions and methods can be used for the therapeutic delivery ofnucleic acids to cells in a subject for the treatment of disease. Theprovided compositions and methods can further be used in scientific,medical or veterinary research. For example, a provided vector systemcan be used to deliver an exogenous nucleic acid to a cell to evaluateits interaction with other molecules in the cell. The provided vectorsystems can, for example, be used to study signal transduction,metabolic pathways, apoptosis, or cell cycle/growth in cells wherein thevector system is used to deliver nucleic acids to either overexpress orinhibit, e.g. by siRNAs, components of these pathways. The providedcompositions and methods can further be used in vaccine production inavian or insect cultures. The provided compositions and methods canfurther be used in the preparation of a medicament for the delivery of anucleic acid to a cell or cell in a subject.

The use of AAVs to deliver genes to the lung by transcytosis would be ofbenefit in genetic diseases like cystic fibrosis,pseudohypoaldosteronism, and immotile cilia syndrome. Furthermore,delivering genes to the lung would be of impact in several non-geneticdiseases. For example, delivering genes that make antibiotic likepeptides to the cells underlying the epithelia would be useful toprevent or treat bronchitis; delivering genes that make growth factorswould be of value in common diseases like chronic bronchitis. Also, AAVscould be used to deliver genes that may play a role in asthma, likeIL-10, or antibodies to IgE and interleukins. The use of an AAV vectorto deliver genes through the alveolar epithelia would be of benefit ingenetic diseases like alpha-1-antitrypsin deficiency. Furthermore,delivering genes through the alveolar epithelia would be of significancein several pulmonary non-genetic diseases. For example, delivering genesthat make antibiotic like peptides would be useful to prevent or treatpneumonia (perhaps of antibiotic-resistant organisms); delivering genesthat make growth factors would be of value in emphysema; deliveringgenes that over-express the epithelial sodium channel or the Na—K ATPasecould be used to treat cardiogenic and non-cardiogenic pulmonary edema;delivering genes that have an anti-fibrosis effect like interferon forpulmonary fibrosis would also be useful. Also, AAVs could be used todeliver genes that may have a systemic effect like anti-hypertensiondrugs, insulin, coagulation factors, antibiotics, growth factors,hormones and others.

The use of AAVs to deliver genes to the central nervous system(CNS)/brain by transcytosis would be of benefit in neurologicaldiseases, including Alzheimer's Disease, Parkinson's Disease,Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia,paranoia, obsessive compulsive disorder, panic disorder, learningdisabilities, ALS, triplet expansions diseases, psychoses, autism,lysosomal storage diseases, Gaucher's disease, Hurler's disease,Krabbe's disease, battens disease, and altered behaviors (e.g.,disorders in feeding, sleep patterns, balance, and perception).

The use of AAVs to deliver genes to the gastrointestinal system/gut bytranscytosis would be of benefit in treatment of diseases and/orGastrointestinal Disorders such as colon cancers, inflammatory boweldisease, diabetes, or Crohn's disease.

The use of AAVs to deliver genes to the genitourinary system bytranscytosis would be of benefit in treatment of diseases of the femalereproductive tract, molecular defects in implantation disorders, andgynecological cancers. These methods would also have contraceptiveapplications.

The use of AAVs to deliver genes to the kidney by transcytosis would beof benefit in treatment of inherited renal disorders such as polycystickidney disease, Alport's syndrome, hereditary nephritis, primaryhyperoxaluria, and cystinuria.

The use of AAVs for wide-spread delivery of genes across blood vesselsinto the muscle would be of benefit in neuromuscular diseases likemuscular dystrophy and Cardiovascular Disorders such as heart disease,restenosis, atherosclerosis, myocarditis, stoke, angina, or thrombosis.

The use of AAVs for wide-spread delivery of genes across blood vesselsinto any/all tissues of a subject would be of benefit in the treatmentof certain cancers (e.g., gastric, ovarian, lung, bladder, liver, andbreast).

The use of AAVs for wide-spread delivery of genes across blood vesselsinto any/all tissues of a subject would be of benefit in the treatmentof certain inflammatory disorders, including, but not limited to,adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis,blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis,cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis,dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis,eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis,gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis,laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis,mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis,orchitis, osteochondritis, otitis, pericarditis, peritendonitis,peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis,pulpitis, retinitis, rhinitis, salpingitis, scleritis,sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis,stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis,and vaginitis; and disorders that are characterized by inflammation suchas hepatitis, rheumatoid arthritis, gout, trauma, pancreatitis,sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave'sdisease, systemic lupus erythematosus, diabetes mellitus, and allogenictransplant rejection.

The use of AAVs for wide-spread delivery of genes across blood vesselsinto any/all tissues of a subject would be of benefit in the treatmentof other diseases, syndromes and conditions, such as adenosine deaminasedeficiency, sickle cell deficiency, thalassemia, hemophilia, diabetes,phenylketonuria, growth disorders, and defects of the immune system.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

Example 1 Isolation, Subcloning, Sequencing and Characterization of BAAV

To understand the nature of BAAV virus and to determine its usefulnessas a vector for gene transfer, it was cloned and sequenced.

Cell Culture and Virus Propagation

293T and COS cells were maintained in DMEM, supplemented with 10% FBS, 2mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cancercell lines indicated in FIG. 1 were cultured in RPMI medium supplementedwith 5% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/mlstreptomycin. MDBK cells were propagated in DMEM supplemented with 5%horse serum, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/mlstreptomycin. Cells were maintained at 37° C. in a 5% CO2 humidifiedatmosphere.

Bovine Adenovirus Type 1 (ATCC VR-313) and Bovine Adenovirus Type 2(ATCC VR-313) obtained from ATCC are reported by ATCC to be contaminatedwith AAV. For virus propagation, MDBK cells were infected with ATCCVR-313 or ATCC VR-314 and cultured for 5 days. At this time, first signsof an adenovirus induced cytopathic effect was observed.

Viral DNA Isolation, Cloning and Sequencing

Viral DNA was isolated from the Bovine Adenovirus Type 1 (ATCC VR-313)and Bovine Adenovirus Type 2 (ATCC VR-313) infected MDBK cells using theHigh Pure Viral Nucleic Acid Kit (Roche). These DNA samples were assayedfor AAV contamination by PCR using the GC Rich PCR Kit (Roche), asdescribed in Katano H, et al. Biotechniques. 2004 April; 36(4):676-80,herein incorporated by reference for its teaching of these methods.Briefly, this method detects the presence of AAV DNA by PCR usingdegenerative PCR primers, which were shown to amplify a fragmentcontaining sequences of the rep and vp ORF of all known AAV serotypes.PCR using DNA isolated from ATCC VR-313 and ATCC VR-314 as templateresulted in the generation of a 1.4 kb amplification product, which wassubsequently cloned using the TOPO TA Cloning KIT (Invitrogen) andsequenced with an ABI Prism 3100 Genetic Analyzer (ABI) and FSdye-terminator chemistry (ABI). The obtained sequences showed homologyto AAV5 rep ORF and AAV4 cap ORF but were not identical to any knownAAV. This result demonstrated that ATCC VR-313 and ATCC VR-314 containedcontaminations of an unknown AAV serotype, termed subsequently bovineadeno-associated virus (BAAV). The obtained sequence of BAAV was used togenerate PCR primers that bind in the BAAV rep ORF in (−) orientationand in the vp ORF in (+) orientation. PCR using these primers andextrachromosomal DNA of ATCC VR-313 infected MDBK cells (isolated usingthe Qiagen Mini Prep Kit) resulted in amplification of a BAAV fragmentspanning from the vp ORF through the ITR to the rep ORF. The PCRamplification products were subsequently cloned using the TA Cloning KIT(Invitrogen) and sequenced with an ABI Prism 3100 Genetic Analyzer (ABI)and FS dye-terminator chemistry (ABI). ITRs of 2 clones were sequencedby isothermal non-cycling sequencing chemistry using radiolabeled dCTP(Epicentre). For the generation of recombinant particles, a BAAVpackaging plasmid was constructed by PCR amplifying a BAAV fragmentcontaining the complete ORF of rep and vp using DNA isolated from ATCCVR-313 and ATCC VR-314 samples as template and inserting this fragmentinto an expression plasmid under the control of a MMTV promoterresulting in the plasmid pMMTV-BAAV#1-200. 10 clones were sequenced. Theplasmids were assayed for the ability to generate recombinant BAAVparticles by transfecting 293 T cells with an AAV5-NLS-GFP vectorplasmid, pMMTV-BAAV and p449b helper plasmid. 2 days after transfection,cells were lysed by 3 freeze thaw cycles. Cleared lysate was used toinfect Cos cells. 2 days after infection, cells were assayed for GFPexpression by fluorescent microscopy. pMMTV-BAAV#47 generated highesttiters of recombinant BAAV but diverged from the BAAV consensus sequenceby 1 nucleotide change. The sequence of pMMTV-BAAV#47 was changed to theconsensus sequence using the Quik Change Kit (Clontech) and namedpMMTV-BAAV.

Sequence Analysis

DNA and protein sequence alignments were performed using the Clustal Wmultiple sequence alignment tool of the Biology Workbench web basedsoftware (SDSC), MacVector 7 (Oxford Molecular). Promoters,transcription initiation and splice sites were predicted using theNeural Network Promoter Prediction web paged software (BDGP). The genomeof BAAV is 4,694 nucleotides in length and has similar organization withthat of other AAVs (FIG. 1A). The entire genome of BAAV displays 54-79%identity at the nucleotide level with the other known AAVs. Highesthomology was observed with AAV5 (79%), lowest homology to BAAV showedAAAV with 54% (FIG. 1B). The BAAV genome has inverted terminal repeatsof 150 nucleotides with are forming the characteristic T-shapedpalindromic structure. The putative Rep-binding element (RBE) consistsof a tandem (GAGY)₄ repeat, and the putative terminal resolution site(trs), AGTGTGG (FIG. 2). The BAAV ITR is greater than 95% identical toAAV5 and contains a trs that is identical to AAV5 as well as a conservedRRE. The Rep ORF of BAAV displays 48-89% identity at the amino acidlevel with the other AAVs, with most of the diversity clustered at theamino termini. A surprisingly high homology of 89% was found with AAV5(FIGS. 3A and 3C). Comparison of the capsid proteins of BAAV and theprimate dependoviruses revealed 55-76% identity with other known AAVs(FIGS. 3B and 3D). AAV4 showed the highest homology to BAAV with 76%while AAAV was most divergent with 55% identity to BAAV Vp1. Divergentregions in the capsid ORF are clustered in surface exposed loops.

Generation of Recombinant Virus

The high homology between the BAAV and AAV5 ITR and Rep amino acidsequence led to the assumption that BAAV can replicate and package AAV5ITR containing vectors. This assumption was confirmed in initialexperiments; AAV5 ITR containing vector plasmids containing a lacZexpression cassette were replicated and packaged with AAV5 or BAAVpackaging plasmids with equal efficiency. Therefore, AAV5 ITR containingvector plasmids were used for all subsequent studies to producerecombinant BAAV.

Recombinant BAAV was generated by transfecting 293 T cells with AAV5vector, BAAV packaging and Ad helper plasmids. 3 confluent T175 flasksof 293T cells were harvested, resuspended in 100 ml DMEM 10% FCS, seededin 10 150 mm plates and incubated at 37° C., 5% CO₂ until cells are 80%confluent (typically 48 h). Cells were transfected with 15 μgpAAV5-NLS-GFP or pAAV5-RnlacZ, 15 μg pMMTV-BAAV and 30 μg p449B. 48 hafter transfection, cells were harvested, washed with PBS andresuspended in 11 ml TD buffer (0.14 M NaCl, 5.0 mM KCl, 0.7 mM K₂HPO₄,25.0 mM Tris, pH7.4. Cells were lysed by 3 freeze thaw cycles andincubated for 30 minutes at 37° C. after adding benzonase to a finalconcentration of 20 U/ml and sodium deoxycholate (final concentration of0.5%). After adding 0.55 g CsCl/ml the lysate was fractionated usingdensity gradient centrifugation in a SW41 rotor for 48 h at 38000 rpm.The gradients were harvested in 0.5 ml aliquots. Aliquots were assayedfor infectivity and particle titer were determined by real time PCRusing primers binding in the promoter region of the vectors.

Determination of Tissue Tropism

Transduction efficiency of recombinant BAAV vector containing anexpression cassette for beta-galactosidase (rBAAV-RnlacZ) was analyzedin 60 cancer cell lines (NCI cancer cell panel). Cells were infectedwith an MOI of 10 with Ad5 and 2 h later with rBAAV-RnlacZ in 10 foldserial dilutions ranging from 10² to 10⁹ particles/well. 48 h afterinfection, cells were fixed and stained for β-galactosidase activitywith 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (GoldBioTechnology, Inc. St. Louis Mo.). Transduced cells were visuallycounted using a light microscope. GFP expressing cells were detectedusing fluorescent microscopy. Results were used to calculate the numberof transduced cells for 10⁹ particles (FIG. 4). rBAAV efficienttransduction of a wide variety of tumor cells including cells of CNS,colon, prostate, renal, breast and ovarian lineage. It is therefore apotent vector for gene transfer in a wide variety of gene therapyapplications.

Due to the high homology between the BAAV and AAV5 ITR as well as Repsequence, it was hypothesized that recombinant BAAV particles carrying alacZ reporter gene or a GFP expression cassette could be produced byco-transfection of AAV5 ITR containing vector plasmids with BAAVpackaging and an adenovirus helper plasmids in 293T cells. Therecombinant particles have a buoyant density in CsCl gradients of 1.375gm/cm³ which is similar to AAV4. These recombinant particles have beenused to compare the transduction efficiency of BAAV with other know AAVisolates and it was found that BAAV has a unique transduction profilecompared to other isolates and is able to transduce a wide variety oftumor cells including cells of CNS, colon, prostate, renal, breast andovarian lineage.

To assess the tropism of BAAV in the lung, primary airway epitheliacells were cultured and plated as previously described (Zabner, J., etal. J Virol. 2000 April; 74(8):3852-8, herein incorporated by referencefor the teaching of these culturing methods) with an equivalent numberof rAAV5 or rBAAV particles containing CMV nuclear GFP (MOI 10) andcultured for over 10 days. GFP expression was determined by flowcytometry (FACS) and the relative transduction was compared. Both AAV5and BAAV efficiently transduced these cells (FIG. 15).

Neutralizing Antibody Assay

AAV isolates that are serological distinct can be distinguished byneutralization assays and are often referred to as AAV serotypes. BAAVwas analyzed to determine if elicits a BAAV-specific immune response inmice that did not cross react with other AAV serotypes. BALB/c mice wereinjected with 10¹⁰ particles of BAAV-RnlacZ, AAV2-RnlacZ, AAV4-RnlacZand AAV5-RnlacZ. 4 weeks after infection serum of the infected animalswas assayed in a neutralizing antibody assay.

Exponentially growing COS cells (7×10³) were plated in a density of7×10³/well in a flat-bottomed 96-well plate. 24 h after seeding, cellswere infected with wild-type adenovirus with a MOI of 10 for 1 h. Heatinactivated sera of rAAV2, rAAV4, rAAV5 and rBAAV infected mice wereserial diluted from 1:200 to a 1:12800 in RPMI containing 1% fatal calfserum (FCS). 40 transducing units of BAAV-RnlacZ (FIG. 5A) orAAV4-RnlacZ (FIG. 5B) (were added to the diluted sera and incubated for1 h at 37° C. Subsequently, the virus/sera mixture was added to COScells. 24 h after rAAV infection, cells were assayed forbeta-galactosidase expression X-Gal staining (Gold BioTechnology, Inc.St. Louis Mo.). Transduced cells were visually counted using a lightmicroscope. Neutralizing titers of the sera ware calculated as thehighest dilution that inhibited 50% of transduction. Any serum dilutionin which more than 70% reduction of positive cells compared withserum-free media remained was considered to be positive for neutralizingactivity. All samples were assayed in duplicate or triplicate.

rBAAV elicited a unique immune response in mice that efficientlyneutralized rBAAV, bud did not cross-react with rAAV4. Sera of rAAV2,rAAV4 and rAAV5 infected mice did not neutralize rBAAV. These resultsdemonstrate that BAAV is a new AAV serotype.

Transduction of Submandibular Glands In Vivo

10¹⁰ particles of AAV2-lacZ and BAAV-lacZ were injected intosubmandibular glands of BALB/c mice by retrograde ductal instillation asdescribed earlier (Yamano et al., 2002). 4 weeks after infection, bloodwas collected form experimental animals by retro-orbital plexus bleed.Submandibular glands were excised, homogenized and lysed in 500 μL ofGalact-light lysis solution (100 mM potassium phosphate (pH7.8), 0.2%TritonX-100) (Applied biosystems). PMSF and leupeptin were added to afinal concentration of 0.2 mM and 5 μg/mL, respectively. The lysate wascleared by centrifugation at 10,000 rpm for 5 min. Genomic DNA wasextracted from a 100-μL aliquot using the Wizard DNA extraction kit(Promega) according to the manufacturer's instructions. DNAconcentrations were determined by spectrophotometry. Detection andquantification of genome copies of the AAV vectors was done byquantitative real time PCR using a TaqMan system (Applied Biosystems)with probes specific to the RSV promoter as described earlier ((Yamanoet al., 2002)). Protein concentration of the lysates was determinedusing the BCA protein assay kit (Pierce) and β-Gal expression wasquantified with a β-Gal ELISA kit (Roche Molecular Biochemicals). Theβ-Gal levels were normalized for total protein concentration andexpressed as picograms of β-Gal per milligram of protein.

Recombinant BAAV was about ten fold more efficient than rAAV2 in thetransduction of submandibular glands and expressing a gene of interest,demonstrating the feasibility of rBAAV to be used as a vector for genetherapy applications (FIG. 6A and FIG. 6B).

BAAV—Non Primate AAV Serotype

Recombinant BAAV has several attributes that make it an attractivevector for gene transfer including unique serological identity, celltropism, and efficient gene transfer in vivo. BAAV is the thirddependovirus of non-primate origin to be cloned and sequenced. The highhomology between BAAV and AAV5 rep along with the biochemically distinctmechanisms of replication for these two viruses compared to othermammalian AAVs suggest that BAAV and AAV5 might form a distinct groupwithin the dependovirus generation. The capsid of BAAV is most similarto that of AAV4, but the divergent regions are clustered mainly on theexposed surface loops that comprise the 3-fold axis of symmetry (aminoacids 429-599 of SEQ ID NO:7). This region is critical for AAV2transduction (Kern, Schmidt et al. 2003) (Opie, Warrington et al. 2003)(Schmidt, Katano et al. 2004). While differences in the capsidbiochemical activity for the different serotypes of AAV are primarilyresponsible for their differences in transduction efficiency, somedifferences may be the result of the ITRs. While AAV2 and AAV4 can bepackaged using AAV2 ITRs, AAV5 and BAAV rep proteins will not initiatereplication of an AAV2 ITR. Therefore, these serotypes were bothpackaged using an AAV5 ITR. However, AAV5 and BAAV, which contain theexact same ITR, but have a significant difference in cell tropism andtransduction efficiency in the inner ear, suggesting that the capsidinteractions are primarily responsible for the differences in serotypetropism.

Example 2 BAAV Efficiently Transduces Neuroepithelial Cells in the InnerEar

This example describes the tropism and transduction efficiency of anovel bovine adenoassociated virus (BAAV) vector in cultured inner earepithelia and compares its infectivity with three, well characterizedprimate adenoassociated vectors: AAV2, -4, and -5. For the first time acytoskeletal protein was used as a reporter gene for viral infection.Beta actin-GFP fusion protein is widely distributed in multiple celltypes and when transiently expressed, it incorporates into hair cellstereocilia and into the apical junctional complex of hair cells andsupporting cells. This example demonstrates that a novel bovine vectorcan efficiently transduce supporting and hair cells of cultured innerear epithelia. Furthermore, prolonged incubation time with viralparticles increases the yield of transduced cells. This novel bovinevirus was significantly more effective in transducing cells of the innerear epithelia than other tested AAV serotypes. Moreover, no pathologicaleffects were demonstrated.

Reagents

Rhodamine/phalloidin and ProLong anti-fade mounting media were fromMolecular Probes (Eugene, Oreg.). Cell Tak was from BD Biosciences (PaloAlto, Calif.). DMEM F-12, L-15 media, fetal bovine serum and ampicillinwere from GIBCO (Carlsbad, Calif.).

Viral Vector Construction

The construction of the beta galactosidase and GFP expression plasmidsis described above. The AAV2 beta actin-GFP fusion expression plasmidwas constructed by subcloning of the CMV-beta-actin-GFP cassette frombeta actin-GFP plasmid (Clontech) into the AAV2 RSV-GFP expressionplasmid and replacement of the RSV GFP cassette with the CMV betaactin-GFP. The AAV5 beta actin-GFP fusion expression plasmid wasproduced in the same manner; however, the CMV beta-actin-GFP cassettewas cloned into the AAV5 RSV-GFP plasmid.

AAV Preparation and Quantification

Recombinant AAV particles were produced by triple transfection of 293 Tcells with AAV helper plasmids expressing the AAV Rep and Cap genes, avector plasmid containing the reporter gene flanked by either type 2ITRs (AAV2, AAV4) or AAV5 ITRs (AAV5, BAAV), and the Ad helper plasmidpAd12 (Smith, Afione et al. 2002). Recombinant vectors were purified byfractionation with CsCl-gradient centrifugation. DNAase resistant genomecopy titers of the vector preparations were determined by quantitativereal time PCR using the TaqMan system (Applied Biosystems) with probesspecific to the RSV promoter. Viruses in CsCl were dialyzed for 24 husing 0.5 ml slide-A-Lyzer (Pierce) in 100 ml of serum free medium withchanging of the medium 3-4 times.

Organotypic Cultures of Rat and Mouse Organ of Corti

Organotypic cultures of rat and mouse organ of Corti and vestibularsensory epithelia were prepared according to a published method(Sobkowicz, Loftus et al. 1993, which is hereby incorporated byreference for its teaching of the method of making organotypic culturesof rat and mouse organ of Corti and vestibular sensory epithelia).Explants from the developing inner ear harvested from neonates can bemaintained for two weeks in culture during which they reach structuraland functional maturity (He, Zheng et al. 2001). PD 0-1 rat pups wereanaesthetized using CO₂ according to NIH guidelines. The skin wascleaned thoroughly with 70% ethanol. After decapitation, both temporalbones were isolated and placed into L-15 media under sterile conditions.Each otic capsule was opened and the stria vascularis, spiral ganglion,Reisner's membrane, and tectorial membrane were removed from all turnsof the cochlea. In addition, the otoconial membrane was removed from themaculae utriculae and saculae. The isolated organ of Corti was dividedfor culturing. Subsequently, the vestibular system was finely dissected.Each sample of the organ of Corti and vestibular system was attached toa Cell Tak-coated coverslip in a culture dish. Cultures were maintainedat 37° C. and 5% CO₂ in DMEM F-12 supplemented with 7% fetal bovineserum containing 1.5 μg/ml ampicillin.

Viral Infection and Histochemistry

Cultured explants of auditory and vestibular sensory epithelia wereinfected with AAV2, AAV4, AAV5, and BAAV viral vectors using betaactin-GFP as a reporter gene (BD Bioscience) in 200 μl of DMEM F-12 at37° C. and 5% CO₂ for the duration of the experiment. Forimmunohistochemistry, cultures were fixed with 4% paraformaldehyde inPBS for 1 h at room temp, permeabilized for 30 min with 0.5% TritonX-100 in PBS, and the actin filaments were counterstained withrhodamine/phalloidin (0.2 U/200 ul Molecular Probes) for 30 min. Stainedexplants were removed from the culture dish and mounted using ProLonganti-fade media. Fluorescence images were obtained either with a ZeissLSM 510 confocal microscope using a 100×1.4 numerical apertureobjective. Image acquisition and post acquisition analysis wereperformed using NIH image and Adobe Photoshop.

Statistical Analyses

Sample frames of sensory epithelia were photographed with a 40×objective. For each measurement, 5 independent frames from at leastthree explanted culture pieces were scored for GFP positive cells andthe total number of cells was determined by scoring the rhodaminephalloidin positive cells. Single factor ANOVA and Student's t testanalyses were performed using Microsoft Excel.

Characterization of BAAV Transduction of Hair Cells

In order to evaluate the tropism of this non-primate bovine vector,BAAV, we incubated cultured explants of rat auditory and vestibularepithelia with BAAV expressing different reporter genes (betagalactosidase, GFP, and beta Actin-GFP). In the preliminary experimentswe used a common reporter gene, GFP. The long, columnar shape of haircells and complex cellular architecture of sensory epithelia, however,made it very difficult to estimate the type and number of transducedcells based only on the diffuse cytoplasmic labeling. To overcome thisdifficulty, we used beta actin-GFP fusion protein as a reporter. Betaactin-GFP can selectively incorporate into hair cells stereocilia(Schneider, Belyantseva et al. 2002)(Rzadzinska, Schneider et al. 2004)as well as into the apical junctional complex of hair cells andsupporting cells. This process allows a straightforward identificationof infected cells on the surface of sensory epithelia.

Hair cell maturation occurs during the first few days after birth.Previous studies have shown that for adenovirus vectors, cell tropismchanged with the maturation of the auditory sensory epithelia (Kanzaki,Ogawa et al. 2002). The tropism and transduction efficiency of BAAV indeveloping (PD 2) and mature (PD 10) inner ear explants was evaluated.Analysis of fixed and counterstained developing cultures after 8 days ofincubation with 10¹⁰ resistant particles/ml (DNAse resistant particles(DRP)/ml) of BAAV revealed transduction of both hair cells andsupporting cells. Inner and outer hair cells of the organ of Corti aswell as vestibular hair cells showed incorporation of beta actin-GFPinto stereocilia starting from their tips. This finding is identical toresults obtained with GeneGun™ plasmid delivery (Schneider, Belyantsevaet al. 2002). We also observed incorporation of beta actin-GFP intoapical junctional complexes of transduced hair cells and supportingcells such as Hensen's, phalangal, interdental, and vestibularsupporting cells. In all of the analyzed explants (n=50) we did notobserve any significant changes in the overall pattern of the reticularlamina even after a prolonged incubation of 8 days with BAAV. Highmagnification images of hundreds of different transduced cells also didnot reveal any signs of structural damage. Because of the cellularcomplexity of inner ear epithelia and the lack of appropriate cellularmarkers, we were unable to determine accurately the total number ofvarious supporting cell types in an explant. We estimated that 100% ofthe supporting Hensen's cells and vestibular supporting cells weretransduced whereas approximately 40% of the phalangal and interdentalcells were transduced. Hair cells were readily quantified by scoringstereocilia bundles and comparing them to the number of phalloidinstained bundles. In PD 2 cultures, BAAV successfully transfected 10% ofinner (n=773) and outer (n=189) hair cells and 48% of vestibular haircells (n=2032). Previous studies suggested that hair cell competencydecreased with the maturation stage of the hair cell (Kanzaki, Ogawa etal. 2002). Therefore, we tested whether the stage of hair cellmaturation influenced BAAV tropism or transduction efficiency.

Qualitative analysis of PD 10 cultures incubated with BAAV revealedsuccessful transfection of hair cells and the same transduction patternobserved in PD 2 cultures. The overall yield of transduced vestibularhair cells in the older cultures was significantly lower (p<0.05) thanin PD 2 explants (17% in PD 10, n=1549 and 48%, n=2032 in PD 2explants). Unfortunately, auditory hair cells progressively degeneratein cultures older than 15 days. Thus, we were unable to estimate numberof infected inner or outer hair cells for these older cultures.Furthermore, the transduction in PD2 cultures was concentrationdependent; increasing concentrations of BAAV vector resulted in asignificant increase in transduction of hair cells (FIG. 7). Thegreatest improvement in transfection yield was observed in vestibularhair cells where almost 50% of the hair cells were transformed after 8days. To analyze if the duration of incubation with viral particlesinfluenced the number of BAAV transduced cells and increased theapparent transfection efficiency, we incubated PD 2 explants with viralparticles for 5 or 8 days (FIG. 8A-B). We observed a significantlyhigher yield (p<0.05) of transduced vestibular and outer hair cellsfollowing a longer incubation time (FIG. 8C). The number of transducedvestibular hair cells increased from 15% on day 5 to 48% after 8 days.Transduction of outer hair cells also increased 4-fold after 8 days butno significant increase of inner hair cells transduction was observed.

This demonstrates for the first time that the present bovineadenoassociated virus vector can efficiently transduce developing andmature hair cells of the organ of Corti and vestibular epithelia as wellas supporting cells of the inner ear explants. The observation thatfunctionally mature hair cells of PD 10 explants can be transformed withBAAV is encouraging and further supports gene transfer using BAAV totransfect hair cells of adult animals.

The observation that the yield of transduced cells increased over timeis consistent with similar observations in vivo AAV5, AAV4 (Davidson,Stein et al. 2000). Interestingly, closer examination of the hairbundles revealed that many of the transduced hair cells incorporatedactin-GFP only at the stereocilia tips. Previous studies showed thatbeta actin-GFP was progressively incorporated into stereocilia startingfrom stereocilia tips as early as 4-6 h after transfection using a genegun. Within 48 to 72 h the entire stereocilia bundle is labeled inauditory and vestibular hair cells respectively. The presence of haircells showing incorporation of beta actin-GFP at the stereocilia tipsafter 8 days of incubation with virus may indicate that the onset ofviral transduction can occur through out the course of experimentalexposure.

The substantial differences in transfection efficiency betweensupporting cells and hair cells prompted us to evaluate the ability ofBAAV to transduce other polarized epithelia. We extended the panel ofepithelial cell lines previously characterized by testing MDCK (dogkidney epithelial cell line) and caco-2 (human adenocarcinoma epithelialcell line) cells because of their overall similarity to inner earsensory epithelia (Schmidt, Katano et al. 2004). Confluent cultures ofMDCK and caco-2 were incubated with BAAV expressing beta actin-GFP atthe concentration 10¹⁰ DRP/ml of viral particles for 8 days.Surprisingly, we did not observe any transduction in MDCK cell cultureseven after 8 days of infection; however, about 20% of caco-2 cellsshowed beta actin-GFP expression.

Comparison of Transduction with Different Serotypes of AAV

Previous studies concluded that AAV2 could transduce cells in the innerear (Luebke, Steiger et al. 2001). Therefore, tropism and transductionefficiency of BAAV was compared in inner ear epithelia with other wellcharacterized serotypes of adenoassociated viruses; AAV2, -4 and -5.Cultured explants of rat auditory and vestibular epithelia (PD2) wereincubated with either AAV2, -4, -5 or BAAV expressing beta actin-GFP ata concentration of 10¹⁰ DRP/ml for 8 days. Confocal analysis of fixedand counterstained samples revealed that overall BAAV was the mosteffective vector for hair cell transduction and supporting cells incultured inner ear sensory epithelia. With BAAV we counted at most 48%of the vestibular, 16% of auditory hair cells, 100% of Hensen's, and 40%of phalangal cells were transfected. On the other hand, culturesincubated with AAV2 showed transduction in 4% of inner hair cells andAAV5 transduced 1% of the vestibular hair cells. Transduction ofsupporting cells with either AAV2 or AAV5 or transduction with AAV4 wasnot observed (FIG. 9). This is in contrast to several studies using AAV2serotypes in adult animals have demonstrated transduction-supportingcells in the inner ear (Li Duan, Bordet et al. 2002). Indeed, we foundthat AAV2 serotypes were much less effective at transducing hair cellsthen BAAV.

Beta Actin-GFP—an Optimal Reporter Gene for Inner Ear Epithelia

Beta actin-GFP used in these studies as a reporter gene for analysis oftropism and infectivity of viral vectors allowed for the identificationof transduced and non-transduced cells in surface preps of sensoryepithelia based on labeling of the hair bundle. In addition,localization of beta actin-GFP into the apical junctional complexes ofhair cells and supporting cells indicated borders between cells in thesecomplex mosaics of different cells and simplified counting of thetransduced cells. Additionally, the ability to follow turnover ofstereocilia actin in cells expressing beta actin-GFP allows for thedetermination of initiation of expression.

Molecular Basis of Specificity of AAV Serotypes

In contrast to BAAV, rAAV2 and rAAV5 were less effective at transducinghair cells. Our in vitro results with rAAV2 are consistent with the invivo studies using AAV2 since less then 2% of the hair cells weretransfected (Luebke, Foster et al. 2001). Efficient AAV2 transductionrequires expression of heparan sulfate proteoglycan on the target cellsurface. Heparan sulfate cytochemistry indicated that hair cells do notexpress this glycoprotein residue on their apical cell surface (Luebke,Steiger et al. 2001). Characterization of the cellular componentsrequired for transduction with AAV4 and AAV5 demonstrate that bothserotypes preferentially bind to 2-3 sialic acid residues but differ intheir linkage specificity. In addition, PDGFRα or PDGFRβ have beenidentified as protein receptors for AAV5 and their expression correlateswith transduction in vivo (Di Pasquale, Davidson et al. 2003), whilesialic acid residues that have been localized to the stereocilia cellsurface are very sparsely distributed on the apical surface of haircells (Suzuki, Katori et al. 1995). Furthermore, only PDGFR alphareceptors have been localized to the lateral wall of vestibular haircells and not the apical surface (Saffer, Gu et al. 1996). Takentogether, these data are consistent with the low transduction efficiencyobserved with primate isolates of AAV2, AAV4, and AAV5.

SUMMARY

BAAV has a 10-fold higher transduction efficiency for neuroepithelialcells of the inner ear as compared to primate derived AAV serotypes.Efficient gene transfer to the cochlea offers both a tool needed for newtherapies for deafness and the ability to study specific genes and theirfunction. The nearly 100% gene transfer in supporting cells is expectedto be useful clinically because many genetic hearing loss diseases arecaused by mutations which effect the supporting cell integrity. Mostimportantly, the availability of a vector, which efficiently transduceshair cells in vivo, advances our ability to characterize the structureand function of the inner ear. The combination of efficiency and lack ofadverse effects makes BAAV an exciting new vector choice for genetransfer to the sensory and nonsensory cells of the inner ear.

Example 3 Role of Sialic Acid and Glycosphingolipids in BAAVTransduction

The role of sialic acid in BAAV transduction was determined by treatingCos with the broad spectrum neuraminidases isolated from V. cholerae,(0.05 U/ml) and a neuraminidase with high specificity for 2-3 sialyllinkages from S. pneumoniae (10 U/ml). 48 h after infection withrecombinant AAV2, AAV4, AAV5, or BAAV expressing GFP, cells wereanalyzed for GFP expression. Neuraminidase treatment resulted inreduction of BAAV transduction (FIG. 10), demonstrating the requirementfor 2-3 linked sialic acid, bound to either a protein or lipid receptorfor BAAV transduction.

The role of glycosphingolipids (GSL) was examined in BAAV mediated genetransfer, by treating Cos cells for 48 h with inhibitors ofglycosphingolipid metabolism, PPMP (5 μM) and PDMP (5 μM), and analyzedthe effect of GSL depletion on BAAV, AAV4 and AAV5 transduction. Weobserved a 90% and 50% inhibition of BAAV mediated gene transferrespectively, whereas AAV4 and AAV5 transduction remained uninhibited(FIG. 11). This implies that the transduction process of BAAV issignificantly different from AAV4 and AAV5, and involves GSLs that acteither as receptors or as essential parts of the uptake machinery.

It was further determined that the receptor for BAAV is proteaseresistant. Cos cells were incubated with 0.025% trypsin or 1 U/mldispase for 30 min. 48 h after infection with recombinant AAV2, AAV4,AAV5 or BAAV expressing GFP, cells were analyzed for GFP expression.Protease treatment resulted in reduction of rAAV2, rAAV4 and rAAV5transduction, while BAAV mediated gene transfer was slightly enhanced(FIG. 12), suggesting that either a protease resistant protein or alipid component is essential for rBAAV binding and uptake.

Example 4 Transcytosis of BAAV Vectors

Previous research had demonstrated that Caco-2 and MDCK cells are modelcell lines for the study of macromolecular transport via transcytosis.Furthermore these cell lines have been used to demonstrate transcytosisof both viruses and proteins. Therefore, to test if AAV can spreadthrough tissue by transcytosis, 2×10⁸ DNA resistant particles ofrecombinant AAV2 (rAAV2) AAV4, AAV5, AAV6, BAAV suspended in 50 ul ofmedium were placed in the upper (apical) side of the transwellpolycarbonate filter over a monolayer of cells each of the followingcells Caco-2, MDCKI, MDCKII, Human primary airways epithelia cells(Airway), Human primary immortalized epithelial endometrial, Bovinebrain primary endothelia cells (BBB), or HeLa. All cultures had TERsindicating the formation of tight junctions and polarized phenotype.After 3 hours of incubation the medium in the basal side of thetranswell was collected and tested for the presence of transcytosed rAAVDNA. Viral DNA was extracted from 200 ul of basal medium and quantifiedby qPCR.

In these cell lines, transcytosis was observed with several AAVserotypes and appeared to be both serotype and tissue-specific (FIG.13). Three hours after the addition of AAV to the apical surface of thecells, over 800,000 particles of AAV5 were present in the media on thebasal lateral side of the trans-well insert of CaCo-2 cells, but not theMDCK, airway epithelia, endometrial, or BBB cells (FIG. 13). SimilarlyBAAV particles were detected in the media on the basal lateral side ofthe MDCK, airways epithelia, endometrial, and BBB cells but not theCaco-2 cells. Interestingly, AAV4 was detected in the basal lateralmedia of all cell types. No virus was detected in the basal lateralmedia when AAV2 was added to the apical surface in either cell type.AAV6 did not transcytose in any of cell types tested, and was not testedon airway epithelia or BBB. HeLa cells do not form barrier epithelia andwere used as a control.

Previous work has demonstrated that transcytosis is a temperaturedependent process than can be inhibited at 4° C. Transcytosis can alsobe inhibited by the addition of agents that selectively fix the plasmamembrane. Recently the addition of tannic acid, a mild fixative agent,to the basal lateral surface blocked the transcytosis of GPI-anchoredproteins to the apical surface (Polishchuk R, Nat Cell Biol. 2004.6(4):297-307). Therefore the ability of this agent to block thetranscytosis of AAV was tested. Treatment of the basal lateral surfaceof either Caco-2 or MDCK cells prior to virus addition to the apicalsurface blocked the accumulation of AAV5 or BAAV in the basal lateralmedia. Furthermore, quantification of the intracellular virusdemonstrated inhibition of exocytosis by tannic acid treatmentdramatically increase the amount of AAV DNA in the cell suggesting theviral particles detected in the basal lateral media are the result of anintracellular transport process and not a paracellular route.

Treatment of the basal lateral surface of Human primary airwaysepithelial cell (HAE) with tannic acid blocked the transcytosis of BAAVor AAV4 vector containing a GFP expression cassette from the apicalsurface to the basal lateral (FIG. 14). Furthermore transductiondramatically increased when assayed at 24 hrs post inoculation. Incontrast no change was observed in AAV2 transduction, which did notdemonstrate any transcytosis activity and has limited binding activityon HAE.

To confirm the DNA detected in the basal lateral media was indeedextracted from intact virus, the material was tested for DNaseresistance after treatment with heat, ionic detergent or protease. Theaddition of DNase alone or in combination with the ionic detergentdeoxycholine had no effect on the viral DNA present in the mediasuggesting it was not free DNA or complexed in lipid vesicles. However,heating to 95° C. prior to treatment with DNAase completely degraded theviral DNA present in the media. This profile is identical to that of theinput AAV particles and suggests the viral DNA is still encapsulated.Titration of the DNase resistant virus in the basal lateral media on Coscells gave a similar particle to infectivity ratio to the input AAVparticles.

While it would appear the AAV DNA detected in the basal lateral media iscontained in intact particles, its presence on the basal lateral surfacecould be the result of lyses of the cells or disruption of themonolayer. Therefore the TER was carefully monitored throughout thecourse of these experiments and was not observed to decrease. To furtherconfirm the integrity of the cell monolayer, mixing experiments werestudied in which two viruses with different gene cassettes were added tothe apical surface at the same time and three hours post addition theamount of each virus in the basal lateral media was quantified usingQPCR specific for each cassette. Both BAAV and AAV5 were able to passfrom the apical to the basal lateral surface of MDCK or Caco cellsrespectively but the AAV2 did not. Therefore the presence of viralparticles in the basal lateral media does not appear to be the result ofa disruption in the cell monolayer.

Taken together this data suggest that dependoviruses particles arecapable of passing through barrier epithelia via transcytosis and theprocess is both serotype and cell type specific.

To further characterize the transcytosis activity observed with AAV5 andBAAV, transcytosis was quantified as both a time and concentrationdependent event. After the addition of particles to the apical surface,samples were removed from the basal lateral media at different timepoints and the amount of virus was quantified by QPCR of the extractedDNA. Viral genomes could be detected as soon as 30 minutes afteraddition and steadily increased with time By 24 hrs, over ⅓ of the inputrecombinant AAV5, BAAVvirus added to Caco or MDCK cells respectively hadbeen transported to the basal lateral surface. In contrast, none of theinput AAV2 or adenovirus was detected on the basal lateral side after 24hrs.

If transcytosis is an activity used by AAV to spread through tissue,this finding would help explain the lack of transduction of barrierepithelia reported with some isolates of AAV. Primary human bronchialairway epithelia (HAE) are known to transport albumin from the apical tothe basal lateral surface by receptor-mediated transcytosis in vivo.While the interaction of BAAV with primary HAE has not beeninvestigated, AAV4, 5 are reported to bind to HAE, however, for AAV4,this interaction does not result in transduction. Because of theinteraction of AAV4 with O-link sialic acid, it was proposed, and hasbeen demonstrated, that mucins, which contained large amounts ofO-linked sialic acid and are expressed on the apical surface of HAE, canblock AAV4 transduction. Alternatively the lack of transduction could bethe result of transcytosis of the virus through the tissue.

To test this hypothesis, AAV2, 4, 5, BAAV were added to the apicalsurface of confluent monolayer cultures of primary human bronchialairway and transcytosis to the basal lateral surface was measured byQPCR after 3 hrs. All cultures had high TERs and expressed ciliatedstructures on their apical surface. Highly differentiated HAE culturesin contrast to immature cultures are resistant to transduction byadenoviral vectors due to a lack of integrin expression that isnecessary for adenovirus entry.

Of the 4 AAVs tested for transcytosis, AAV4 and BAAV were detected inthe basal lateral media. No transport of AAV2 or AAV5 was detected. As acontrol, adenovirus also was tested for transcytosis activity in the HAEcultures, but no transport was detected.

Epithelial cells that line the genitourinary tract form an importantepithelial barrier layer and can transport proteins by transcytosis.AAV2, 4, 5 or BAAV were therefore tested to determine for the ability topenetrate this barrier epithelial layer by transcytosis. Awell-characterized model of endometrial cells has been reported by Kyoet al. Following addition of the 4 AAVs to the apical surface, BAAV andAAV4 could be detected in the basal lateral media when assayed at 3 hrspost inoculation (FIG. 13).

Most AAVs were identified originally as contaminants of laboratorystocks of adenovirus, thus our understanding of their natural biology,cell tropism, and knowledge the cellular components required for virusentry is limited. For AAV5, in addition to N-linked sialic acid, theplatelet derived growth factor (PDGF) receptors were identified asprotein receptors for AAV5 (Di Pasquale et al., Nat Med. 2003 October;9(10):1306-12). This interaction was confirmed by modulation of PDGFRexpression by transfection of expression plasmids, inhibitor treatment,or competition experiments with the extracellular domain of PDGFRα.Likewise AAV5 transduction could be blocked with sialolactosamineconjugates kaludov et al 2001.

Previous research had demonstrated that transcytosis is actin dependentand occurs by a caviolin mediated pathway. Furthermore transcytosis canbe blocked by treatment with tannic acid. Therefore to bettercharacterize the transcytosis pathway utilized by AAV5 in Caco cells thecells were treated with a panel of agents known to block eithertranscytosis in other systems or AAV5 mediated transduction. It wasnoted that AAV5 transcytosis could be inhibited by filipin and nocozodolas well as treatment with tannic acid.

Caco cells, which actively transcytosis AAV5, are not reported toexpress PDGFR and are not transduced by AAV5. In agreement, competitionexperiments with sPDGFRa had little effect on AAV5 transcytosis.Furthermore, competition experiments with 200 ug/ml sialolactosamine or200 ug/ml heparin did not inhibited AAV5 transcytosis.

Both BSA and transferrin are reported to transcytosis through Caco cellsvia distinct receptor mediated pathways. However competition with eitheragent did not inhibit AAV5 transcytosis suggesting the AAV5 could use adistinct pathway.

In addition to confirming the intracellular nature of AAV5 transcytosisin Caco cells, the above experiments suggest that AAV5 transcytosis isoccurring by a pathway independent of the one described fortransduction. To confirm this Caco cells were stably transfected withPDGFRa and assayed for both transcytosis and transduction activity. Cacocells were not permissive for AAV5 transduction, however transductiondramatically increase following stable expression of PDGFRa. In contrastonly a minor increase in transcytosis activity was detected in theCaco/PDGFRa cells.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

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1-108. (canceled)
 109. An isolated nucleic acid molecule comprising anucleic acid sequence encoding a polypeptide selected from the groupconsisting of: a) a polypeptide comprising a unique fragment of an aminoacid sequence selected from the group consisting of SEQ ID NO:3 and SEQID NO:5; b) a polypeptide comprising an amino acid sequence at least 80%identical over the entire length of an amino acid sequence selected fromthe group consisting of SEQ ID NO:3 and SEQ ID NO:5.
 110. The isolatednucleic acid molecule of claim 109, wherein the encoded polypeptide hasat least one activity selected from the group consisting of a) bindingan antibody selective for a polypeptide consisting of SEQ ID NO:3 or SEQID NO:5; and b) binding to the Rep binding site of an AAV genome. 111.The isolated nucleic acid molecule of claim 109, wherein the encodedpolypeptide comprises: i) at least one epitope from a sequence selectedfrom the group consisting of SEQ ID NO:3 and SEQ ID NO:5; ii) at least40 contiguous amino acids from a sequence selected from the groupconsisting of SEQ ID NO:3 and SEQ ID NO:5; or, iv) at least 100contiguous amino acids from a sequence selected from the groupconsisting of SEQ ID NO:3 and SEQ ID NO:5.
 112. The isolated nucleicacid molecule of claim 109, wherein the amino acid sequence of theencoded polypeptide differs from an amino acid sequence selected fromthe group consisting of SEQ ID NO:3 and SEQ ID NO:5 as a result of oneor more conservative substitutions.
 113. The isolated nucleic acidmolecule of claim 109, wherein the encoded polypeptide comprises: i) anamino acid sequence at least 90% identical over the entire length of anamino acid sequence selected from the group consisting of SEQ ID NO:3and SEQ ID NO; and, ii) an amino acid sequence at least 95% identicalover the entire length of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3 and SEQ ID NO:5.
 114. The isolated nucleicacid molecule of claim 109, wherein the encoded polypeptide comprises asequence selected from the group consisting of SEQ ID NO:3 and SEQ IDNO:5.
 115. The isolated nucleic acid molecule of claim 109, wherein thenucleic acid sequence comprises: i) at least 30 contiguous nucleotidesfrom a sequence selected from the group consisting of SEQ ID NO:2 andSEQ ID NO:4.
 116. The isolated nucleic acid molecule of claim 109,wherein the nucleic acid sequence comprises a polynucleotide sequence atleast 90% identical to a sequence selected from the group consisting ofSEQ ID NO:2 and SEQ ID NO:4.
 117. The isolated nucleic acid molecule ofclaim 109, wherein the nucleic acid sequence comprises a sequenceselected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.